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The hairy roots developed after Agrobacterium rhizogenes transformation can be used for the production of secondary metabolites which show medicinal properties. Plant secondary metabolites include various classes of chemical compounds, such as the tropane alkaloids found in some members of the Solanaceae family, for example, Atropa belladonna, Datura stramonium, D. innoxia, Hyoscyamus muticus, and H. niger. This chapter presents the strategy for producing tropane alkaloids such as scopolamine and hyoscyamine by the hairy roots of these species. High biomass production of D. stramonium and D. innoxia hairy roots and the associated bioactive compounds can be enabled by optimization of the medium. In addition, scopolamine and hyoscyamine production can be enhanced by optimizing various culture conditions, such as the type of the medium or sucrose concentration, and the ploidy level of H. muticus hairy roots. Their production by A. belladonna hairy roots can also be influenced by the ammonium or nitrate concentration of the medium. Tropane alkaloid production by hairy roots can be significantly increased by the use of elicitor treatments such as chitosan, acetic acid, citric acid, jasmonic acid, acetylsalicylic acid, or aluminum chloride: for example, treatment with Tween 20, combined with L-phenylalanine or DL-s-phenyllactic acid alone or with tropinone feeding, increased the level of hyoscyamine released into the medium by D. innoxia hairy roots. Secondary metabolite production can also be improved by transgenic manipulation: tropane alkaloid biosynthesis can be raised by overexpression of the genes encoding the enzymes involved in the tropane alkaloid biosynthesis pathway such as hyoscyamine-6s-hydroxylase (H6H), putrescine N-methyltransferase (PMT), or tropinone reductase I and II (TR I and TR II). Alkaloid production can be influenced by overexpression of genes encoding enzymes involved in the tropane alkaloid biosynthesis pathway, as well as by genetic manipulation based on gene silencing. The cultivation of D. innoxia and H. niger hairy roots in bubbling bioreactors may also serve as an efficient culture for large-scale tropane alkaloid production.

Six previously undescribed tropane alkaloids, designated as erythrobezerrines A-F, were isolated from the EtOH extract from the stem bark of Erythroxylum bezerrae Plowman. Their structures were elucidated based on the interpretation of the NMR and MS data and in some instances, confirmed by X-ray diffraction analysis. The cytotoxicity of the isolated compounds was evaluated against the cancer cell lines L929, PC-3, HCT-116, SNB-19 and NCI-H460, but only erythrobezerrine C showed moderate activity with IC50 values of 3.38 and 5.43 μM for HCT-116 and NCI-H460, respectively.

A novel method was developed and applied to the determination of the most representative tropane alkaloids (TAs), atropine and scopolamine, in gluten-free (GF) grains and flours by HPLC-MS/MS. Accordingly a suitable sample treatment procedure based on solid-liquid extraction (SLE) and followed by strong cation-exchange solid-phase extraction (SCX-SPE) was optimized. SBA-15 mesostructured silica functionalized with sulfonic acids was evaluated as sorbent. The proposed method was fully validated in sorghum flour showing good accuracy with recoveries in the range of 93&ndash, 105%, good linearity (R2 &gt, 0.999) and adequate precision (RSD &lt, 20%). Low method quantification limits (MQL) were obtained (1.5 and 2.4 &micro, g/kg for atropine and scopolamine, respectively) and no matrix effect was observed thanks to the extraction and clean-up protocol applied. The method was applied to 15 types of GF samples of pseudocereals (buckwheat, quinoa and amaranth), cereals (teff, corn and blue corn, sorghum and millet) and legumes (red and green lentil, chickpea and pea). Atropine was found above the MQL in eight of them, with values between 7 and 78 &micro, g/kg, while scopolamine was only found in teff flour, its concentration being 28 &micro, g/kg. The method developed is an interesting tool for determining TAs in a variety of samples of GF grains and flours.

Research on Duboisia plants as the main source of the tropane alkaloids scopolamine and hyoscyamine started in the 1940s. In this way, basic knowledge about the plants and their cultivation became available, which was the prerequisite for domestication, commercial production, and breeding of high-yielding Duboisia genotypes. All approaches had the aim to increase tropane alkaloid yield through improvement of tropane alkaloid productivity within plant material.

A wide-scope analytical method was developed and validated for the determination of tropane alkaloids (TAs) in honey samples. A simple and fast extraction procedure, using a mixture of methanol/water/formic acid (75/25/0.4, v/v/v) as extraction solvent, followed by a clean-up with graphitized black carbon (GBC) and magnesium sulphate was optimized, and compounds were analysed by liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS-Orbitrap). Validation of the proposed method provided adequate linearity (R2 > 0.99), trueness (recoveries 71–120%) and precision (relative standard deviation, RSD ≤ 20.1%), with limits of quantitation (LOQs) at 20 µg/kg (except anisodamine and scopolamine at 40 µg/kg) and a significant matrix effect (≤−50%). Nineteen honey samples were analysed, but only one was positive, containing 27 µg/kg of scopolamine. Additionally, a post-targeted screening was performed, and 47% of samples were contaminated with different herbicides, insecticides and veterinary drugs. Therefore, the proposed analytical method is a powerful tool for both targeted TAs and post-targeted contaminant analyses.

The application of ionic liquids (ILs) for the separation of bioactive compounds from various sample matrices is a burgeoning area. Here, a class of porous materials called metal–organic frameworks (MOFs) were combined with tropine-based ILs having structural similarity with natural tropane alkaloids for the quantitative analysis of Huashansheng tablets. Three MOFs (MIL-101, HKUST-1 and ZIF-8) were used to form the composite with the a tropine-based IL (N-propyl-substituted tropine hexafluorophosphate, [C3tr][PF6]), using the ship-in-a-bottle (SIB) synthesis approach. The performance of the hybrid IL/MOF composite (IL@MOF) was evaluated as a potential sorbent for application in the dispersive solid-phase-extraction-based enrichment of tropane alkaloids, followed by chromatographic analysis. It was found that [C3tr][PF6]@MIL-101 exhibited excellent adsorption capacity, and the extraction results showed satisfactory recoveries in the range of 91.5–104.7% (RSD < 5%). Further, the limit of detection went down to 10–20 μg L−1 with a linear range from 100–500 μg L−1, which proved that an adsorbent with the same nucleus as the target objects would be more suitable and efficient for their later extraction.

The genus Erythroxylum contains species used by indigenous people of South America long before the domestication of plants. Two species, E. coca and E. novogranatense, have been utilized for thousands of years specifically for their tropane alkaloid content. While abuse of the narcotic cocaine has impacted society on many levels, these species and their wild relatives contain untapped resources for the benefit of mankind in the form of foods, pharmaceuticals, phytotherapeutic products, and other high-value plant-derived metabolites. In this review, we describe the current state of knowledge of members within the genus and the recent advances in the realm of molecular biology and biochemistry.

In this research, tropane alkaloids in Radix physochlainae were extracted by tropine-type ionic liquid (IL) aqueous solutions under ultrasound assistance, and N-propyltropine hexafluorophosphate ([C3Tr][PF6]) was found to be the most ideal IL in this extraction mode after comprehensive screening. When 0.03 mol/L [C3Tr][PF6] aqueous solution was chosen as the extraction solvent, the solid-liquid ratio of raw material powders and ionic liquid aqueous solution was 1:20 (g/mL), ultrasonic power was 90 W and extraction time was 30 min, the extraction efficiency of tropane alkaloids has reached 121.3%. Compared with common heating extraction, it can further shorten the extraction time, improve extraction efficiency and decrease IL consumption. Furthermore, extraction mechanism together with potential toxicity of IL have been explored and discussed.

Tropane alkaloids (TAs) are a class of phytochemicals produced by plants of the nightshade family used for treating diverse neurological disorders. Here, we demonstrate de novo production of tropine, a key intermediate in the biosynthetic pathway of medicinal TAs such as scopolamine, from simple carbon and nitrogen sources in yeast (Saccharomyces cerevisiae). Our engineered strain incorporates 15 additional genes, including 11 derived from diverse plants and bacteria, and 7 disruptions to yeast regulatory or biosynthetic proteins to produce tropine at titers of 6 mg/L. We also demonstrate the utility of our engineered yeast platform for the discovery of TA derivatives by combining biosynthetic modules from distant plant lineages to achieve de novo production of cinnamoyltropine, a non-canonical TA. Our engineered strain constitutes a starting point for future optimization efforts towards realizing industrial fermentation of medicinal TAs and a platform for the synthesis of TA derivatives with enhanced bioactivities., Tropane alkaloids (TAs) are a group of phytochemicals that are used to treat neurological disorders. Here, the authors engineer baker’s yeast to produce tropine, a key intermediate in the biosynthetic pathway of TAs, and cinnamoyltropine, a non-canonical TA, from simple carbon and nitrogen sources.

Alkaloids are specialized metabolites produced by plants to protect themselves from biotic and abiotic forces. Tropane and granatane alkaloids are of special importance due to their vast pharmacological benefits. The evolutionary origin of the biosynthesis of these alkaloids is unknown. Understanding this aspect of tropane and granatane alkaloid biosynthesis will help us gain a greater understanding of what is necessary to produce these alkaloids using a systems biology approach. Tropane alkaloids are produced amongst seven different plant families; and granatane alkaloids are produced amongst four different families. The biosynthesis of tropane alkaloids is well studied in the Solanceae family. Here I report an alternate route to tropane alkaloid biosynthesis in the Erythroxylaceae, which utilizes a spermidine methyltransferase versus the well-studied putrescine methyltransferase found in the Solanaceae. I also provide evidence for a homologous step in granatane alkaloid biosynthesis using a novel N-methyltransferase called cadaverine methyltransferase. With the discovery and investigation of novel N-methyltransferases involved in both tropane and granatane alkaloid biosynthesis, we can utilize these enzymes to produce these compounds using a metabolic engineering platform in a microbial or plant system.

Datura innoxia Mill., a traditional Chinese herbal medicine, produces tropane alkaloids such as hyoscyamine and scopolamine. Scopolamine has a larger demand than hyoscyamine due to its stronger pharmacological effects and fewer side reactions. It is extracted from solanaceous plants. However, the content of scopolamine is lower than hyoscyamine in D. innoxia. Hyoscyamine 6β-hydroxylase (H6H, EC1.14.11.11) is the key enzyme which can catalyze hyoscyamine to form scopolamine. In this study, a cDNA encoding H6H was cloned from D. innoxia roots and named Dih6h. The full-length cDNA is 1413 bp in length with a 1044-bp open reading frame encoding 347 amino acids. The deduced protein sequence of D. innoxia H6H (DiH6H) shared high identity with H6Hs from other plants. The DiH6H was heterologously expressed in Escherichia coli and purified via His-tag affinity technique. The recombinant DiH6H showed activity in transforming hyoscyamine to scopolamine. Despite Dih6h mRNA was detected in various tissues, its levels in roots were higher than that in other tissues. Indeed, scopolamine accumulation was low in roots, but it was very high in aerial parts, especially in flowers and seeds. These observations suggest that scopolamine may be synthesized in the roots and subsequently transported to the aerial parts. To further verify in vivo function of DiH6H, the cDNA of DiH6H was overexpressed in D. innoxia hairy roots. As expected, an increase of scopolamine production was observed in the positive transformants. The results provide a potential strategy for increasing scopolamine yield by metabolic engineering of its biosynthetic pathway in D. innoxia.

A high-affinity anti-cocaine monoclonal antibody, designated h2E2, is entering phase 1 clinical trials for cocaine abuse therapy. To gain insight into the molecular details of its structure that are important for binding cocaine and cocaine metabolites, the Fab fragment was generated and crystallized with and without ligand. Structures of the unliganded Fab and the Fab fragment bound to benzoylecgonine were determined, and were compared with each other and with other crystallized anti-cocaine antibodies. The affinity of the h2E2 antibody for cocaine is 4 nM, while that of the cocaine metabolite benzoylecgonine is 20 nM. Both are higher than the reported affinity for cocaine of the two previously crystallized anti-cocaine antibodies. Consistent with cocaine fluorescent quenching binding studies for the h2E2 mAb, four aromatic residues in the CDR regions of the Fab (TyrL32, TyrL96, TrpL91 and TrpH33) were found to be involved in ligand binding. The aromatic side chains surround and trap the tropane moiety of the ligand in the complex structure, forming significant van der Waals interactions which may account for the higher affinity observed for the h2E2 antibody. A water molecule mediates hydrogen bonding between the antibody and the carbonyl group of the benzoyl ester. The affinity of binding to h2E2 of benzoylecgonine differs only by a factor of five compared with that of cocaine; therefore, it is suggested that h2E2 would bind cocaine in the same way as observed in the Fab–benzoylecgonine complex, with minor rearrangements of some hypervariable segments of the antibody.

Tropine and pseudotropine with opposite stereospecific configurations as platform compounds are central building blocks in both biosynthesis and chemical synthesis of pharmacologically important tropane and nortropane alkaloids. The supply of plant-derived tropine and pseudotropine still heavily depends on either plant extraction or chemical synthesis. Advances in synthetic biology prompt the microbial synthesis of various valuable chemicals. With the biosynthetic pathway elucidation of tropine and pseudotropine in several Solanaceae plants, the key genes were sequentially identified. Here, the enzymes responsible for converting N-methylpyrrolinium into tropine and pseudotropine from Anisodus acutangulus were characterized. Reconstruction of the six-step biosynthetic pathways into Saccharomyces cerevisiae provides cell chassis producing tropine and pseudotropine with 0.13 and 0.08 mg/L titers from simple feedstocks in a shake flask, respectively. The strains described not only offer alternative sources of these central intermediates and their derived alkaloids but also provide platforms for pathway enzyme discovery.

Molecularly imprinted polymers (MIPs) prepared using conventional functional monomers exhibit poor specific extraction of scopolamine from tropane alkaloids, which hinders their application in separation and purification. In this paper, a novel molecularly imprinted polymer (MIP) was prepared by precipitation polymerization using scopolamine as the template, monoethyl fumarate (MFMA) as a functional monomer, and ethylene dimethacrylate (EGDMA) as a cross-linker. The advantages of the supercritical fluid technology for the removal of the template were verified by comparing the efficiency of the swelling method and the Soxhlet extraction method. The prepared MFMA-based MIPs (MFMA-MIPs) showed a high adsorption capacity (49.75 mg g−1) and high selectivity toward scopolamine with a selectivity coefficient of 3.5. 1H NMR spectroscopy was performed to demonstrate the interactions between the two functional groups of the functional monomer and the template. Lastly, MFMA-MIPs were used as solid phase extraction (SPE) sorbents for scopolamine analysis. It was found that 97.0–107.0% of the template had been extracted using the SPE column from the complex of scopolamine, atropine and anisodamine. The mean recoveries of scopolamine from plant samples were 96.0–106.0% using the established method, which showed a good linearity in the range of 8.0–4.0 × 104 μg L−1. The results showed that MFMA-MIPs could be applied for the separation of scopolamine from tropane alkaloids.

International audience; The present study describes the isolation of convolvine from the roots of the Tunisian plant Convolvulus dorycnium L. and it's synthesis through a four-steps sequence starting from tropine. Then, an efficient synthesis of N-(het)aryl tropanes derivativesby a sequence of a palladium-catalyzed N-arylationof convolvine has been established. This strategyenabled an access to unknown tropane scaffolds of biological interests.

Dopamine transporter (DAT) and glucose metabolism imaging have been applied in the diagnosis of Parkinson’s disease (PD). We explored the possibility of evaluating for PD with NeuroQ software by analyzing 11C-2β-carbomethoxy-3β-(4-fluorophenyl) tropane (11C-CFT) and 18F-FDG PET/CT. We retrospectively analyzed brain 11C-CFT and 18F-FDG PET/CT of 38 patients with parkinsonism, including 20 with PD, 10 with multiple system atrophy (MSA) and 8 with essential tremor (ET), and compared them with the PET/CT of 11 normal healthy controls (NC). PD patients were divided into mild and moderate-severe grade according to the Hoehn-Yahr (H&Y) scale. The 11C-CFT uptake in the caudate nuclei (CN) and putamen (Pu) normalized with cerebellum (CN/Cb and Pu/Cb) were obtained with a manual method and NeuroQ software, and their diagnostic performance was compared.18F-FDG uptake of specific regions was also obtained with NeuroQ, and the enhancement effect for the differential diagnosis was evaluated. There was significant agreement between the manual method and the NeuroQ method for 11C-CFT uptake by CN (r2= 0.680) and Pu (r2= 0.770). 11C-CFT uptake by CN and Pu in PD and MSA patients was significantly lower compared to NC and ET patients. The cutoffs of CN/Cb and Pu/Cb for the distinction between PD and NC were 1.71 and 2.20, respectively. No difference in uptake ratios occurred between PD and MSA. 18F-FDG uptake by the pons and cerebellum in the MSA group was markedly decreased. It was highly accurate in distinguishing between PD and MSA when combined with analysis of 11C-CFT uptake. Pu/Cb decreased significantly in mild grade PD compared to NC group (1.92 ± 0.33 vs. 2.82 ± 0.43); however no statistically significant decrease in CN/Cb was observed until moderate-severe grade PD (1.43 ± 0.11 vs. 2.23 ± 0.36). In early asymmetric PD, a statistically significant difference could be seen with Pu/Cb between the symptomatic and asymptomatic side (2.17 ± 0.30 vs. 1.95 ± 0.22). 11C-CFT and 18F-FDG PET/CT can be analyzed quantitatively with NeuroQ software, which provides an accurate method for the diagnosis and severity evaluation of PD.

Tropane alkaloids are toxic secondary metabolites found in agricultural weeds, like jimson weed (Datura stramonium) and henbane (Hyoscyamus niger). The intake of food and feed contaminated with tropane alkaloids led to several incidents of poisoning in humans and animals. Because data on the transfer of tropane alkaloids into the milk of exposed dairy cows were not available in scientific literature, the present study was carried out. Four cows received tropane alkaloids (atropine sulfate and scopolamine hydrobromide trihydrate) orally in three increasing dosage levels for five days each. The total doses of pharmacologically active alkaloids (sum of l -hyoscyamine and scopolamine) at the levels 1, 2, and 3 were 93, 186, and 279 μg/kg body weight/day, respectively. None of the applied dosage levels induced obvious clinical symptoms in the cows. The alkaloid content in composite milk of individual cows was measured for each milking time. Even though the maximum mean transfer rates (atropine: 0.037%, scopolamine: 0.007%) were very low, a transfer of tropane alkaloids into milk could already be proven at the lowest dosage level. At the highest dosage level, the biologically active substances were detected in milk at a mean level of 1.60 ± 0.07 μg/kg. The results obtained in this study indicate that under particular circumstances contaminated raw milk may contribute to the exposure of consumers to tropane alkaloids.

A simple, sensitive, selective, and rapid optical nanobiosensor based on FRET was designed to detect tropane alkaloids as anti-cholinergic agents in natural and transgenic hairy roots extracts of Atropa belladonna. To achieve that, conjugation of tioglycolyic acid capped cadmium telluride quantum Dots, M2 muscarinic receptor (Cd/Te QDs-M2R) and conjugation of scopolamine-rhodamine123 (Sc-Rho123) were performed. More specifically, proportional amounts of M2 muscarinic receptor and quantum dots (QDs) were conjugated while scopolamine (as a tropane alkaloid) and rhodamine123 were also combined and these moieties functioned as donor and acceptor pairs, respectively. The system response was linear over the range of 0.01-4µmolL-1 of scopolamine hydrochloride concentration with a detection limit of 0.001µmolL-1. The developed nanobiosensor was successfully used for in vitro recognition of scopolamine as an anti-cholinergic agent in the investigated plant extracts. In addition, Agrobacterium rhizogenesis mediated gene transfer technique was employed to generate hairy roots and to enhance the production of tropane alkaloids in the studied medicinal plant.

Tropane alkaloids are toxic secondary metabolites produced by Solanaceae plants. Among them, plants from Datura genus produce significant amounts of scopolamine and hyoscyamine; the latter undergoes racemization to atropine during isolation. Because of their biological importance, toxic properties and commonly reported food and animal feed contamination by different Datura sp. organs, there is a constant need for reliable methods for the analysis of tropane alkaloids in many matrices. In the current study, three extraction and sample-clean up procedures for the determination of scopolamine and atropine in plant material were compared in terms of their effectiveness and repeatability. Standard liquid-liquid extraction (LLE) and EXtrelut® NT 3 columns were used for the sample clean-up. Combined ultrasound-assisted extraction and 24h static extraction using ethyl acetate, followed by multiple LLE steps was found the most effective separation method among tested. However, absolute extraction recovery was relatively low and reached 45-67% for atropine and 52-73% for scopolamine, depending on the compound concentration. The same method was also the most effective one for the isolation of target compounds from Datura stramonium leaves. EXtrelut® columns, on the other hand, displayed relatively low effectiveness in isolating atropine and scopolamine from such a complex matrix and hence could not be recommended. The most effective method was also applied to the extraction of alkaloids from roots and stems of D. stramonium. Quantitative analyses were performed using validated method based on gas chromatography with flame ionization detector (GC-FID). Based on the results, the importance of the proper selection of internal standards in the analysis of tropane alkaloids was stressed out.

Marco Contoli,1 Paolo Solidoro,2 Fabiano Di Marco,3,4 Nicola Scichilone,5 Angelo Corsico,6 Fulvio Braido,7 Pierachille Santus4,8 1Research Centre on Asthma and COPD, Department of Medical Sciences, University of Ferrara, Ferrara, Italy; 2Cardiovascular and Thoracic Department, Città della Salute, Turin, Italy; 3Department of Health Sciences, University of Milan, Milan, Italy; 4Respiratory Unit, San Paolo Hospital, Milan, Italy; 5Department of Internal Medicine, Section of Pulmonology (DIBIMIS), University of Palermo, Palermo, Italy; 6Department of Molecular Medicine, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; 7Allergy and Respiratory Diseases Clinic, DIMI, University of Genoa, IRCS AOU San Martino-IST, Genoa, Italy; 8Pulmonary Rehabilitation Unit, Fondazione Salvatore Maugeri, Scientific Institute of Milan-IRCCS, Milan, Italy Abstract: The pathophysiology of chronic obstructive pulmonary disease (COPD) includes persistent airflow limitation, altered gas exchange, and enhanced chronic inflammatory response. According to disease severity in individual patients, exacerbations and comorbidities frequently occur. The overall nocturnal and daily symptoms have a strong impact on patient quality of life and clinical outcomes. Bronchodilators, by targeting two important aspects of COPD pathophysiology, ie, bronchoconstriction and lung hyperinflation, are the mainstay of therapy for COPD. Aclidinium bromide in particular is an anticholinergic molecule, approved for maintenance bronchodilator treatment of stable COPD, that combines high antimuscarinic activity with strong kinetic selectivity for the M3 receptor subtype. Moreover, the elevated plasma clearance of aclidinium has been related to low systemic bioavailability and low incidence of anticholinergic adverse events, whereas the reduced residence time at M2 receptors provides good cardiovascular safety. Altogether, these characteristics result in a high safety and tolerability profile. This review aims to reappraise the contribution of symptoms and of the level of quality of life determinants on COPD severity and to evaluate how therapeutic strategies with aclidinium may positively impact on these specific determinants of disease severity. Keywords: COPD, quality of life, daily symptoms, LAMA, aclidinium

Duboisia species, which belong to the family of Solanaceae, are commercially cultivated in large scale, as they are main source of the pharmaceutically-used active compound scopolamine. In this study, 1 H NMR-based metabolite profiling linking primary with secondary metabolism and additional quantification via HPCL-MS with special focus on the tropane alkaloids were applied to compare leaf and root extracts of three wild types and two hybrids of Duboisia myoporoides and D. leichhardtii at different developmental stages grown under controlled conditions in climate chambers and under agricultural field plantation. Based on the leaf extracts, a clear distinction between the Duboisia hybrids and the wild types Duboisia myoporoides and D. leichhardtii using principal component analysis of 1 H NMR data was observed. The average content in scopolamine in the hybrids of Duboisia cultivated in climate chambers increased significantly from month 3–6 after potting of the rooted cuttings, however not so for the examined wild types. The Duboisia hybrids grown in climate chambers showed higher growth and contained more sugars and amino acids than Duboisia hybrids grown in the field, which in contrast showed an enhanced flux towards tropane alkaloids as well as flavonoids. For a more detailed analysis of tropane alkaloids, an appropriate HPLC-MS method was developed and validated. The measurements revealed large differences in the alkaloid pattern within the different genotypes under investigation, especially regarding the last enzymatic step, the conversion from hyoscamine to scopolamine by the hyoscyamine 6β–hydroxylase. Scopolamine was found in highest concentrations in Duboisia hybrids (20.04 ± 4.05 and 17.82 ± 3.52 mg/g dry wt) followed by Duboisia myoporoides (12.71 ± 2.55 mg/g dry wt), both showing a high selectivity for scopolamine in contrast to Duboisia leichhardtii (3.38 ± 0.59 and 5.09 ± 1.24 mg/g dry wt) with hyoscyamine being the predominant alkaloid.

Tropane alkaloids and their derivatives are anticholinergic drugs with narrow therapeutic range. Here we characterize the organic cation transporters from the SLC22 (OCT1, OCT2, and OCT3) and the SLC47 families (MATE1 and MATE2-K) as potential mediators of the renal and extra-renal excretion, the two major roads of elimination of these substances. All analyzed compounds inhibited and the quaternary amine derivatives ipratropium and trospium were strongly transported by OCTs and MATEs. Overexpression of OCTs or MATEs in HEK293 cells resulted in an up to 63-fold increase in the uptake of ipratropium (K m of 0.32 μm to OCT2 and V max of 3.34 nmol×mg protein−1×min−1 to MATE1). The transcellular transport of ipratropium was 16-fold higher in OCT2-MATE1 and 10-fold higher in OCT1-MATE1 overexpressing compared to control MDCKII cells. Genetic polymorphisms in OCT1 and OCT2 affected ipratropium uptake and clinically relevant concentration of ondansetron and pyrithiamine inhibited ipratropium uptake via MATEs by more than 90%. This study suggests that OCT1, OCT2 and MATEs may be strongly involved in the renal and extra-renal elimination of ipratropium and other quaternary amine alkaloids. These substances have a notoriously narrow therapeutic range and the drug-drug interactions suggested here should be further critically evaluated in humans.

A chiral-NHC-catalyzed highly diastereo- and enantioselective dearomatizing double Mannich reaction of isoquinolines was developed that provides a powerful and straightforward synthetic route toward substituted tropane derivatives with four contiguous stereocenters. A unique feature of this strategy is the use of readily available isoquinolines to provide two reactive sites for dearomatization, thus opening up an unprecedented approach to tropane derivatives with excellent stereoselectivity. The four-component reactions proceeded smoothly with good results. Thus, the present method is suitable for the diversity-oriented synthesis of useful tropane derivatives with high efficiency.

A procedure is proposed for determining some narcotic and psychoactive substances (cathinones, tropane alkaloids, derivatives of ?-aminobutyric acid) in urine, including sample preparation and the determination of analytes by HPLC coupled with electrospray ionization tandem mass spectrometry. Sample preparation involved acid hydrolysis or the dilution of the sample. The lower limit of quantification is 200 pg/mL; the limit of detection is 100 pg/mL; and the calibration curves are linear in the range 0.2–50 ng/mL. The procedure was tested on real samples provided by the Toxicological Department of the Krasnodar Regional Narcological Dispensary. The high sensitivity of the analytical system ensures its use for the determination of narcotic preparations within a certain time after their intake, which may be of particular importance for forensic examinations.

Background Hyoscyamus albus L. ( Solanaceae ) an old medicinal plant is a rich source of tropane and nortropane alkaloids which confers to this plant a number of very interesting and beneficial therapeutic effects. Purpose Calystegines that are polyhydroxylated alkaloids and imino-sugars poccess significant glycosidases inhibitory activities and are therefore good candidats for the treatment of diabetes mellitus. Study design Calystegines extracted from Hyoscyamys albus seeds were tested for teir acute oral toxicity and investigated for their in-vivo antidiabetic effect on Streptozotocine induced diabetes in mice. Methodes Calystegines were extracted from the seeds plant using an Ion exchange column; the remaining extract was then administrated orally to mice at several single doses for acute toxicity assay. A dose of 130 mg/kg streptozotocine was injected to mice to induce diabetes mellitus, and diabetic mice were treated orally during 20 days with 10 mg/kg and 20 mg/kg calystegines and 20 mg/kg glibenclamide as the reference drug. Results Acute oral toxicity showed that calystegines are not toxic up to a dose of 2000 mg/kg with absence of any signs of intoxication and damages in Liver and kidney tissues. The nortropane alkaloids markedly reduced blood glucose levels and lipid parameters of diabetic mice to normal concentrations after 20 days of treatment at 10 mg/kg and 20 mg/ kg ( p 0.05). Histopathological study of diabetic mice pancreas indicated that calystegines of Hyoscyamus albus have minimized streptozotocine damages on β-cells of islets of langerhans, stimulated β-cells regeneration and improved with this insulin secretion. Conclusion The findings of this study suggest that calystegines are potent antidiabetic agents with antihyperglicemic and hypolipidemic effects, and a protective fonction on pancreas in streptozotocin induced diabetes in mice.

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.

Treehoppers (Hemiptera: Membracidae) are sap-feeding insects distributed mainly in tropical regions. Alchisme grossa is a treehopper that has been reported in the Bolivian Yungas forests using mostly Brugmansia suaveolens (Solanaceae) as host-plant, where adult females oviposit and take care of their nymphs until they molt to adults. Brugmansia is a subtropical genus producing a variety of tropane alkaloids (TAs). We herein report the sequestration by adult males and females of A. grossa of TAs from B. suaveolens, examining separately the distinct body sections of insects. Purified extracts of A. grossa and B. suaveolens were analyzed by gas chromatography/mass spectrometry. TAs in A. grossa were the same as those in its host-plant; furthermore, they were equally distributed between sexes and they were differentially allocated within the body of adult individuals. An ecological role for sequestered TAs is discussed.

Tropane alkaloids are bioactive metabolites with great importance in the pharmaceutical industry and the most important class of natural products found in the Erythroxylum genus. However, these compounds are usually separated by traditional chromatographic techniques, in which the sample is progressively purified in multiple chromatographic steps, resulting in a time- and solvent-consuming procedure. In this work we present the isolation of a novel alkaloid, 6β,7β-dibenzoyloxytropan-3α-ol, together with the two known 3α-benzoyloxynortropan-6β-ol and 3α,6β-dibenzoyloxytropane alkaloids, directly from the crude alkaloid fraction from the leaves of Erythroxylum subsessile, by using a single run pH-zone-refining counter-current chromatography method. The ethyl acetate/water (1:1, v/v) biphasic solvent system with triethylamine and HCl as retention and eluter agents, respectively, was used to isolate tropane alkaloids for the first time. The structures of the isolated alkaloids were elucidated by spectroscopic methods.

Atropa belladonna L. is one of the most important herbal plants that produces hyoscyamine or atropine, and it also produces anisodamine and scopolamine. However, the in planta hyoscyamine content is very low, and it is difficult and expensive to independently separate hyoscyamine from the tropane alkaloids in A. belladonna. Therefore, it is vital to develop A. belladonna plants with high yields of hyoscyamine, and without anisodamine and scopolamine. In this study, we generated A. belladonna plants without anisodamine and scopolamine, via the CRISPR/Cas9-based disruption of hyoscyamine 6β-hydroxylase (AbH6H), for the first time. Hyoscyamine production was significantly elevated, while neither anisodamine nor scopolamine were produced, in the A. belladonna plants with homozygous mutations in AbH6H. In summary, new varieties of A. belladonna with high yields of hyoscyamine and without anisodamine and scopolamine have great potential applicability in producing hyoscyamine at a low cost.

The alkaloid extract of datura stramonium (rich of atropine, hyoscyamine, scopolamine) is used to evaluate its effect on the central nervous system in rats (locomotion, anxiety). The animals received the alkaloid extract of datura by intraperitoneal injection. However, after the injection a group of rats got an inhibitory treatment contain flavonoids extracted from Rosmarinus officinalis. At the dose of 50 mg/kg/body weight of the alkaloid extract, the female rats have shown a reduction of locomotor activity and an induction of an anxiogenic effect. In the other hand, the mixture of Datura stramonium and Rosmarinus officinalis showed a neuroprotective action with regard to the disorders induced by extracts of Datura stramonium.

Monitoring of tropane alkaloids is regulated in the European Union in cereal-based foods for infants and young children, tea and herbal infusions. The European Commission’s Joint Research Centre (JRC) developed an improved LC-MS/MS analytical method using a pentafluorophenyl column, validated it and conducted two proficiency tests targeting these food categories. A subset of the data gathered from laboratories that used the JRC method was additionally exploited to derive interlaboratory performance characteristics. The method showed fit-for-purpose figures of merit. The LOQs for atropine and scopolamine were around 0.4 and 1.2 µg/kg in cereal products, and in tea and herbal infusions, respectively. Uncertainties varied from 15 to 25%. The reproducibility varied from 11 to 38% for scopolamine and from 17 to 44% for atropine at levels ranging from 0.18 to 18.8 and 1.2–54.0 µg/kg, respectively. Recoveries ranged from 71 to 96%. These performance parameters render the method a good candidate for standardisation.

Successful production of medicinal compounds from plants depends on weather and growing seasons, potentially making supply chains vulnerable to uncontrollable forces. One way around such challenges...

Since ancient civilization, plants have been utilized in many aspects of life, especially in medicinal purposes due to the presence of distinctive secondary metabolites like alkaloids, phenolics and terpenoids. Among them, alkaloids represent a large group of secondary metabolites that have basic properties and comprise nitrogen atom within the heterocyclic ring. Plant synthesizes alkaloids to maintain their survivability under unfavorable conditions. Over 3000 years, indigenous people have been used alkaloid-containing plant extracts to treat several ailments such as fever, snakebite and insanity. However, despite significant benefits to humans and pharmaceutical industries, some of the plant alkaloids are categorized as main plant toxins due to their enormous structural diversity and various modes of actions. Humans and animals can be exposed to toxic alkaloids either through inhalation, swallowing or by direct contact, therefore leads to the specific mechanism that involves receptors, transporters, enzymes and genetic materials at specific cells and tissues, hence may cause hepatotoxic effects and musculoskeletal deformities. This review focuses on some of the plant alkaloids such as pyrrolizidine, tropane, piperidine and indolizidine, which can give various side effects on humans and animals such as itching, nausea, vomiting, mild gastrointestinal perturbation, psychosis, paralysis, teratogenicity, arrhythmias and sudden death.

Tropane alkaloids (TA) are valuable secondary plant metabolites which are mostly found in high concentrations in the Solanaceae and Erythroxylaceae families. The TAs, which are characterized by their unique bicyclic tropane ring system, can be divided into three major groups: hyoscyamine and scopolamine, cocaine and calystegines. Although all TAs have the same basic structure, they differ immensely in their biological, chemical and pharmacological properties. Scopolamine, also known as hyoscine, has the largest legitimate market as a pharmacological agent due to its treatment of nausea, vomiting, motion sickness, as well as smooth muscle spasms while cocaine is the 2nd most frequently consumed illicit drug globally. This review provides a comprehensive overview of TAs, highlighting their structural diversity, use in pharmaceutical therapy from both historical and modern perspectives, natural biosynthesis in planta and emerging production possibilities using tissue culture and microbial biosynthesis of these compounds.

A hairy root clone (M8) of Atropa belladonna, producing high levels of tropane alkaloids, was established by transformation with Agrobacterium rhizogenes (MAFF 03-01724). Littorine, an intermediate of tropane alkaloids, was detected by high-performance liquid chromatography and gas chromatography-mass spectrometry in the alkaloid fraction of the hairy roots and identified by nuclear magnetic resonance analysis. Littorine was also detected in the non-transformed root culture of A. belladonna.

We present a case of suspected idiopathic Parkinson's disease in whom extrastriatal accumulation of Technetium-99m labeled tropane derivatives in a pituitary macroadenoma interfered with image quality and interpretation. Subsequent F-18 fluoro-dihydroxyphenylalanine positron emission tomography/computed tomography was useful to demonstrate the absence of presynaptic dopaminergic dysfunction.

Parkinson&rsquo, s disease (PD) is a neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta, leading to alteration of the integrity of dopaminergic transporters (DATs). In recent years, some radiopharmaceuticals have been used in the clinic to evaluate the integrity of DATs. These include tropane derivatives such as radiolabeled &beta, CIT and FP-CIT with iodine-123 (123I), and TRODAT-1 with metastable technetium-99 (99mTc). Radiolabeling of &beta, CIT with radioactive iodine is based on electrophilic radioiodination using oxidizing agents, such as Chloramine T or Iodo-Gen&reg, For the first time, the present work performed a comparative study of the radiolabeling of &beta, CIT with iodine-131 (131I), using either Chloramine T or Iodo-Gen&reg, as oxidizing agents, in order to improve the radiolabeling process of &beta, CIT and to choose the most advantageous oxidizing agent to be used in nuclear medicine. Both radiolabeling methods were similar and resulted in high radiochemical yield (&gt, 95%), with suitable 131I-&beta, CIT stability up to 72 h. Although Chloramine T is a strong oxidizing agent, it was as effective as Iodo-Gen&reg, for &beta, CIT radiolabeling with 131I, with the advantage of briefer reaction time and solubility in aqueous medium.

Alkaloids represent a very large and chemically inhomogeneous group of nitrogen compounds isolated from different natural sources. They have long been studied due to their specific toxicological characteristics, as some of them are suspected of having very dangerous properties, but there has still been little investigation of the existence of possible glycoside precursors in plant products. An analytical method based on a suspect screening approach using liquid chromatography coupled with high resolution tandem mass spectrometry in neutral loss (NL) mode was used for the tentative identification of alkaloid precursors. This NL approach applied to 15 plant extracts made it possible to tentatively identify 41 glycosidic precursors (4 monopentosides, 3 monodeoxyhexosides, 24 monohexosides, 1 pentoside-hexoside and 9 dihexosides) in 6 of them, belonging to the piperidine (21), pyrrolizidine (9), isoquinoline (7), indole (3) and tropane (1) alkaloid groups. This work confirms the promising investigative ability of a suspect screening approach in this complex field of study.

Theories of plant invasion based on enemy release in a new range assume that selection exerted by specialist herbivores on defence traits should be reduced, absent, or even selected against in the new environment. Here, we measured phenotypic selection on atropine and scopolamine concentration of Datura stramonium in eight native (Mexico) and 14 non‐native (Spain) populations. Native populations produced between 20 and 40 times more alkaloid than non‐native populations (atropine: 2.0171 vs. 0.0458 mg/g; scopolamine: 1.004 vs. 0.0488 mg/g, respectively). Selection on alkaloids was negative for atropine and positive for scopolamine concentration in both ranges. However, the effect sizes of selection gradients were only significant in the native range. Our results support the assumption that the reduction of plant defence in the absence of the plant's natural enemies in invasive ranges is driven by natural selection.

Summary The undemanding LC-MS/MS method was developed for the synchronized analysis of atropine and scopolamine in maize crop. The dSPE was carried out with 1% acetic acid in acetonitrile/water and a mixture of magnesium sulphate, sodium chloride and sodium citrate. The analytes were separated on a Zorbax XDB C18 column using methanol/water as the mobile phase in gradient mode. The detection was done using a tandem masss spectrometry (MS/MS) in the positive ESI. The tropane alkaloids exhibited excellent linearity in the range of 2-20 µg/kg with the LOQ of 5 µg/kg for maize. The extraction recoveries of atropine and scopolamine were 65.7 and 85.5% with the intraday RSDr 10.25 and 4.29%, respectively. The validated method was appled to real maze samples. One sample contained 18.8 µg/kg of atropine and 6.3 µg/kg of scopolamine.

Tropanes are an important class of alkaloid natural products that are found in plants all over the world. These compounds can exhibit significant biological activity and are among the oldest known medicines. In the early 19th century, tropanes were isolated, characterized, and synthesized by notable chemical researchers. Their significant biological activities have inspired tremendous research efforts toward their synthesis and the elucidation of their pharmacological activity both in academia and in industry. In this chapter, which addresses the developments in this field since 1994, the focus is on the synthesis of these compounds, and several examples of sophisticated synthetic protocols involving both asymmetric and catalytic approaches are described. In addition, the structures of more than 100 new alkaloids are included as well as the applications and pharmacological properties of some tropane alkaloids.

Hyoscyamus reticulatus L. is a herbaceous biennial belonging to the solanaceae family. Hyoscyamine and scopolamine as main tropane alkaloids accumulated in henbane are widely used in medicine to treat diseases such as parkinson’s or to calm schizoid patients. Hairy roots media manipulation which uses elicitors to activate defense mechanisms is one of the main strategies for inducing secondary metabolism as well as increasing the production of valuable metabolites. Cotyledon-derived hairy root cultures were transformed by Agrobacterium rhizogenes. Sodium nitroprusside (SNP), a nitric oxide donor), was used in various concentrations (0, 50, 100, 200 and 300 uM) and exposure times (24 and 48 h). Treatment with SNP led to a significant reduction in fresh and dry weight of hairy roots, compared to control cultures. ANOVA results showed that elicitation of hairy root cultures with SNP at different concentrations and exposure times significantly affected the activity of as antioxidant enzymes such as catalase (CAT), per-oxidase (POD) and ascorbate peroxidase (APX). The highest hyoscyamine and scopolamine production (about 1.2-fold and 1.5-fold increases over the control) was observed at 50 and 100 uM SNP at 48 and 24 hours of exposure time, respectively. This is the first report of SNP elicitation effects on the production of tropane alkaloids in hairy root cultures.

Anticholinergic drugs based on tropane alkaloids, including atropine, scopolamine, and hyoscyamine, have been used for various medicinal and toxic purposes for millennia. These drugs are competitive antagonists of acetylcholine muscarinic (M-) receptors that potently modulate the central nervous system (CNS). Currently used clinically to treat vomiting, nausea, and bradycardia, as well as alongside other anesthetics to avoid vagal inhibition, these drugs also evoke potent psychotropic effects, including characteristic delirium-like states with hallucinations, altered mood, and cognitive deficits. Given the growing clinical importance of anti-M deliriant hallucinogens, here we discuss their use and abuse, clinical importance, and the growing value in preclinical (experimental) animal models relevant to modeling CNS functions and dysfunctions.

Scopolia tangutica is a traditional Chinese medicine used for antispasmodic, anesthesia, analgesia, and sedation. Its medicinal activity is associated to alkaloid constituents, including tropane and cinnamamide types. Low content of alkaloids in plant makes them difficult to be isolated and identified. The present work developed an effective method to quickly characterize alkaloids from Scopolia tangutica by high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry. Thirteen reference compounds were studied for their fragmentation pathways, including five tropane alkaloids and eight cinnamamide ones. Alkaloid constituent was analyzed by an optimized high-performance liquid chromatography method and mass spectrometry analysis to achieve systematic characterization of alkaloids from Scopolia tangutica. As a result, 53 compounds were identified, including 21 tropane alkaloids (eight new ones), 18 caffeoyl ones (ten new ones) and 14 dicaffeoyl ones (seven new ones). It was important to provide rich information in phytochemical study and structure-guided isolation of important compounds from this plant.

ATAD2 is a cancer-associated protein whose bromodomain has been described as among the least druggable of its class. In our recent disclosure of the first chemical probe against this bromodomain, GSK8814 (6), we described the use of a conformationally constrained methoxy piperidine to gain selectivity over the BET bromodomains. Here we describe an orthogonal conformational restriction strategy of the piperidine ring to give potent and selective tropane inhibitors and show structural insights into why this was more challenging than expected. Greater understanding of why different rational approaches succeeded or failed should help in the future design of selectivity in the bromodomain family.

Emulsion liquid membrane (ELM) coupled with tropine-based ionic liquid was prepared and successfully adopted for the extraction and quantitative analysis of tropane alkaloids (TA) in Radix physochlainae. Effects of formation conditions of ELM were explored and then optimized; the ideal oil-water mass ratio was determined to be 1:2.5, chloroform, Span 80 and 0.05 mol L−1 N-propyl-tropine hexafluorophosphate ([C3Tr][PF6]) aqueous solution were stirred for 30 min with the speed of 1500 r min-1. In order to extract and enrich TA with ELM efficiently, key factors related with their extraction efficiency such as stirring speed, volume of the extracts (feed solution), migration time and initial concentration of TA were systematically investigated. Under optimal conditions, 1.5 mL extracts containing 1.6 mg mL−1 of TA was stirred with ELM at 250 r min-1 for 5 min, the extraction efficiency of target alkaloid can reach 94.14%. Finally, the method could be used in quantitative analysis of TA in herbal material and patent medicine after demulsification. By comparison, application of ELM for the enrichment and quantitation of TA offers a more straightforward and effective strategy, which is expected to provide a meaningful reference for similar separation processes.