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Anisodus tanguticus (Maxim.) Pascher is an important Tibetan folk medicine and the source of tropane alkaloids (TAs) grown in Qinghai-Tibet Plateau. There are marked differences in quality of A. tanguticus from geographic areas. The aim of present research was to establish a method for the quantitative analysis of TAs coupled with chemometrics analysis to trace geographical origins. Qualitative analysis of TAs in A. tanguticus was carried out using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry and quantitative analysis of TAs in different plant organs from different geographical origin was achieved. Contents of TAs were subjected to the principal component analysis, and orthogonal partial least-squares discriminant analysis. The contents of the three marker compounds (anisodamine, anisodine and atropine) in the roots and acrial parts of A. tanguticus were positive correlated and varied significantly from different geographical origins. Principal component analysis, and orthogonal partial least-squares discriminant analysis results showed excellent discrimination between different geographical origin of A. tanguticus. This study could provide comprehensive evaluation and further utilization of A. tanguticus resources.

Covering: 2015 to 2020Nitrogen heterocyclic natural products (NHNPs) are primary or secondary metabolites containing nitrogen heterocyclic (N-heterocyclic) skeletons. Due to the existence of the N-heterocyclic structure, NHNPs exhibit various bioactivities such as anticancer and antibacterial, which makes them widely used in medicines, pesticides, and food additives. However, the low content of these NHNPs in native organisms severely restricts their commercial application. Although a variety of NHNPs have been produced through extraction or chemical synthesis strategies, these methods suffer from several problems. The development of biotechnology provides new options for the production of NHNPs. This review introduces the recent progress of two strategies for the biosynthesis of NHNPs: enzymatic biosynthesis and microbial cell factory. In the enzymatic biosynthesis part, the recent progress in the mining of enzymes that synthesize N-heterocyclic skeletons (e.g., pyrrole, piperidine, diketopiperazine, and isoquinoline), the engineering of tailoring enzymes, and enzyme cascades constructed to synthesize NHNPs are discussed. In the microbial cell factory part, with tropane alkaloids (TAs) and tetrahydroisoquinoline (THIQ) alkaloids as the representative compounds, the strategies of unraveling unknown natural biosynthesis pathways of NHNPs in plants are summarized, and various metabolic engineering strategies to enhance their production in microbes are introduced. Ultimately, future perspectives for accelerating the biosynthesis of NHNPs are discussed.

Tropane alkaloids, including clinically important hyoscyamine and scopolamine, are produced in the roots of medicinal plant species, such as Atropa belladonna, from the Solanaceae family. Recent molecular and genomic approaches have advanced our understanding of the metabolic enzymes involved in tropane alkaloid biosynthesis. A noncanonical type III polyketide synthase, pyrrolidine ketide synthase (PYKS) catalyzes a two-step decarboxylative reaction, which involves imine–ketide condensation indispensable to tropane skeleton construction. In this study, we generated pyks mutant A. belladonna hairy roots via CRISPR/Cas9-mediated genome editing and analyzed the metabolic consequences of the loss of PYKS activity on tropane alkaloids, providing insights into a crucial role of the scaffold-forming reaction in the biosynthetic pathway.

Datura metel (Solanaceae) which is commonly known as thorn’s apple, Indian apple or devil’s trumpet isan annual herb of temperate zones which is distributed all over the world. D. metel belongs to the family solanaceae. From a longer period of time (37 A.D), species of this family had therapeutic uses. This article is based on the review of different scientific backgrounds and studies regarding the traditional uses, phytochemistry, biochemical constituents and pharmacological uses of D. metel. This review is based on the facts of the available literature review. Different researchers conducted researches and studies on D. metel and confirmed the presence of enormous chemical compounds like flavonoids, tropane alkaloids, tannins, saponins and withanolides. D. metel has been found to be pharmacologically important species because of its different pharmacological and traditional uses, such as hepatoprotective, antiviral effect, antibacterial effect, anti-asthmatic, analgesic, antipyretic and nephroprotective effect, anticancer and antifungal effect. However, further in vivo and in vitro advanced studies are required to carried out for the exact pharmacological mechanisms and for basis of clinical utility.

Seven new tropane alkaloids, including five monomeric (1-5), one dimeric (6), and one trimeric (7) 3α-nortropane ester, along with two known monomeric nortropane alkaloids (8 and 9), were isolated from the leaves and bark of Pellacalyx saccardianus. Their structures, including the absolute configuration of the enantiomeric pair of (±)-6, were elucidated by comprehensive spectroscopic analyses. Alkaloids 6 and 7 showed cytotoxicity toward human pancreatic cancer cell lines (AsPC-1, BxPC3, PANC-1, and SW1990). Alkaloids 1, 4, and 9 induced a smooth muscle relaxation effect comparable to that of atropine (Emax 106.1 ± 7.5%, 97.0 ± 5.2%, 100.9 ± 1.4%, 111.7 ± 1.7%, respectively) on isolated rat tracheal rings.

Piper sarmentosum Roxb. (PS) and Piper cathayanum M. G. Gilbert & N. H. Xia (PC) are both listed as Traditional Chinese Medicine herbs. In this study, ultra-performance liquid chromatography mass spectrometry (UPLC-MS/MS) was used to analyze secondary metabolites and metabolically profile the leaves, stems and roots in PS and PC. A total of 110 and 92 differential metabolites were shared from various organs of PS and PC, respectively. Compared to the stems and roots of PS or PC, alkaloids and phenols in leaves of PS or PC accounted for 70% and 57% of the total alkaloids and phenols, respectively. An analysis of enriched metabolic pathways demonstrated that L-tyrosine and 4-hydroxycinnamic acid in PS were directly involved in five and three metabolic pathways, which are related to the biosynthesis of isoquinoline alkaloids and phenol backbones, respectively. The L-glutamic acid and oxoglutaric acid in PC were involved in seven and two metabolic pathways, respectively. L-glutamic acid synthesizes heme, tropane, piperidine and pyridine alkaloids, and the type Ⅱ polyketide backbone. Moreover, oxoglutaric acid generated via L-glutamic acid was mainly involved in the citrate cycle and played a fundamental role in determining these important metabolic processes.

Datura stramonium L. (Solanaceae) possesses a rich tropane alkaloids (TAs) spectrum. The plant contains, in particular, the allelopathic compounds scopolamine and atropine, which are poorly soluble in water, thus limiting their use in agrochemical formulations as biocidal and deterrent agents against herbivore insects. The efficacy of the hydrophobic TAs extracts could be increased with the improvement of their dissolution/leaching properties. This is important for improving screening and test performance and for elucidating the activity of environmentally friendly agricultural approaches, with new perspectives for the production and use of those biodegradable insecticidal products. The present study explores the aspects of atropine and scopolamine complexation with cyclodextrin (CDs) through FT-IR and UV–Vis spectroscopies. In addition, the structures of the inclusion complex of atropine, scopolamine and β-CD have been investigated by molecular modeling techniques. The results obtained indicate that β-CDs are a promising carriers for improving the properties of TAs, therefore increasing their application potential in agrochemical formulations. Graphic abstract

Spices, herbs, and tea have been used for dietary purposes since ancient times. Some of them may contain toxic natural plant toxins synthesized in plant secondary metabolism or because of co-harves...

Pharmacologically important tropane alkaloids such as hyoscyamine, and scopolamine, are synthesized in the roots of several Solanaceae and also accumulate in their shoots. The tropane alkaloids content in roots of D. innoxia seedlings was almost 3-fold higher than in their shoots. Moreover, scopolamine accounted for 77 % of the total tropane alkaloid content in shoots as compared to only 20 % in roots. All candidate genes in the scopolamine biosynthetic pathway were significantly expressed in D. innoxia mature roots with limited expression in mature leaves. Using exogenous isotopically labeled 2H5 L-phenylalanine, we show here that root tissues, but not detached shoots, readily biosynthesize scopolamine. Tropane alkaloids newly synthesized by roots, and preferentially scopolamine, were further transported to shoots. Our evidence indicates that D. innoxia seedlings have the ability to synthesize tropane alkaloids in their roots and differentially transport them to the shoots.

The [3 + 2]-cycloaddition reaction between N-tosylaziridines and α,β-unsaturated ketones was promoted with lithium iodide. The reaction proceeded under mild conditions to provide N-tosylpyrrolidines. Quaternary carbon-possessing 3,3-disubstituted pyrrolidines including spiro compounds were afforded in high yields. A simple procedure with easy to handle reagents makes this reaction concise. The intramolecular version of this reaction was applied to synthesize tropane skeletons.

Climate-driven invasions of toxin-producing plants compromise human health, food safety, and food security. A recent poisoning that involved cereal products distributed by the World Food Programme revealed contamination with tropane alkaloids from seeds of invasive common thorn-apple. With continued global change, plant toxin contaminations could become a more widespread phenomenon.

Summary Alkaloids and alkaloid-producing plants have the potential to reduce crop damage by plant-parasitic nematodes. In a series of in vitro experiments, the nematicidal activity of the tropane alkaloids, hyoscyamine and scopolamine, and a mixture of both on the root-knot nematode, Meloidogyne incognita, was tested. Solutions of 16 mg of compound per ml of solvent were used in concentrations of 80-1280 μg ml−1 of water. Inactivity of second-stage juveniles of M. incognita increased with increasing concentration and exposure time. Lethal concentrations (LC50) after 120 min of exposure ranged from 182.4 μg ml−1 for scopolamine to 318.4 μg ml−1 for hyoscyamine and 332.8 μg ml−1 for the combination of both alkaloids. Similarly, the same concentrations of scopolamine inhibited hatching of M. incognita to a greater extent than hyoscyamine. In a glasshouse experiment, M. incognita was not able to penetrate the roots, induce galls and reproduce on Datura stramonium, D. innoxia and D. tatula. Results indicate that the tropane alkaloids hyoscyamine and scopolamine contained in Datura plants express a strong nematicidal activity against M. incognita and could possibly be used for an alternative and sustainable nematode management.

Hyoscyamine, a member of the class of compounds known as tropane alkaloids (TAs), is clinically used as an anticholinergic drug. Previous research has predicted that hyoscyamine is produced via the...

A new tropane alkaloid 7β-acetoxy-6β-benzoyloxy-3α-hydroxytropane (1) was isolated from the twigs of Erythroxylum macrocalyx Mart. (Erythroxylaceae), along with the known substances: 6β,7β-dibenzoyloxy-3α-hydroxytropane (2), 6β,7β-dihydroxy-3α-(phenylacetoxy)tropane (3), 3α-benzoyloxy-6β,7β-dihydroxytropane (4), 6β-benzoyloxy-3α-(4-hydroxy-3,5-dimetoxybenzoyloxy)tropane (5), ombuin-3-rutinoside-5-glucoside (6), lupeol (7), taraxerol (8) and lupenone (9). Compounds 1 and 2 were also isolated from the leaves. The structures were established by analyses of 1D- and 2D-NMR and MS data, as well as by comparison with literature data for known compounds. The structure of 2 was also supported by X-ray crystallography analyses. The compounds were evaluated in vitro for their antibacterial and antiproliferative activities. Compound 5 showed high antiproliferative activity on liver hepatocellular carcinoma cells (HepG2) with IC50 value of 3.66 μg mL−1 (8.29 μmol L-1), but no cytotoxic effect (IC50 > 25 μg mL-1) on human lymphoblast cell line. This study reveals the potential use of 5 as prototype for the synthesis of new antiproliferative agents.

Plant alkaloids, are one of the largest groups of pharmaceutical metabolites. Scopolamine and atropine are most common tropane alkaloids in Atropa belladonna L. with great application in pharmaceutical industry. Phytochemical biosynthetic pathways induction via various elicitors is an effective strategy for increasing the production of valuable substances. Therefore, in this research effect of yeast extract were studied on expression of h6h gene, antioxidant enzymes activity and production of tropane alkaloid in A. belladonna hairy roots. Hairy roots of A. belladonna were obtained from cotyledonary leaf explants by A7 strain of Agrobacterium rhizogenes. Polymerase chain reaction (PCR) analysis with specific primers confirmed the insertion of rolB in putative transgenic hairy roots genome. Different concentrations of yeast extract (YE) (0, 0.5, 1 and 1.5 mg/L) at different exposure time (24 and 48 h) were used as elicitor. Scopolamine and atropine content and activity of antioxidant enzymes such as guaiacol peroxidase, ascorbate peroxidase and catalase increased and expression of h6h (hyoscyamine 6 beta-hydroxylase) gene in elicited hairy roots were investigated. High concentration of YE decreased the growth of hairy roots but increased the atropine content compared to the control. According to the results, the highest content of scopolamine (9.21%) and atropine (43.39%) which were 9 and 5 fold than control, were obtained by 1 and 1.5 mg/L YE at 48 h exposure time, respectively. The results demonstrated that YE can be used as effective elicitor to increase tropane alkaloids production in hairy root cultures of A. belladonna.

Hyoscyamine, anisodamine and scopolamine are tropane alkaloids present in some Solanaceae species and used in modern medicine. L-Hyoscyamine is hydroxylated to 6β-hydroxyhyoscyamine (anisodamine) and then epoxidated to scopolamine by the dual action of hyoscyamine 6β-hydroxylase (H6H), a 2-oxoglutarate dependent dioxygenase. A natural mutation in the Gly-220 residue to Cys was previously shown to be associated with the loss of function of H6H in Mandragora officinarum, preventing the accumulation of anisodamine and scopolamine in these plants. We show here that a deliberate Gly220Cys mutation in the Datura innoxia DiH6H protein caused a loss of both its enzymatic abilities and rendered it unable to hydroxylate L-hyoscyamine into anisodamine and to epoxidate anisodamine into scopolamine. By using protein modeling based on an available crystal structure of H6H from Datura metel, we show how the Cys220 residue causes a steric interference in the active site cavity impairing the interaction of both substrates, hyoscyamine and anisodamine with the active site of the protein. We also address the enantiomeric preference of DiH6H based on molecular modeling.

This paper evaluates the occurrence of the TAs in 71 food products samples. The analysis comprised the following samples: 18 corn puffs, 12 popcorn, 16 corn and 25 corn grits samples, obtained from the shops in Belgrade and Novi Sad during 2019. and 2020. The overall number of the samples with the atropine and scopolamine detections below the LOQ was 61,97%. The percentage of the detection of the tropane alkaloids above the limit of quantification, which may have a negative effect on the health of humans and animals, was 29,57%. The atropine and scopolamine detections are equal in the number of corn puffs, popcorn and corn samples, while 7 samples of corn grits contained the atropine, and 6 of them contained the scopolamine. The highest mean concentrations of the atropine and scopolamine were detected in corn grits, followed by popcorn, then in the corn, while the lowest were in the corn puffs.

The development of radiometal‐labelled pharmaceuticals for neuroimaging could offer great potential due to easier handling during labelling and availability through radionuclide generator systems. Nonetheless, to date, no such tracers are available for positron emission tomography, primarily owing to the challenge of crossing the blood–brain barrier (BBB) and loss of affinity through chelator attachment. We have prepared a variety of 68Ga‐labelled phenyltropanes showing that, through a simple hydrocarbon‐linker, it is possible to introduce a chelator onto the lead structure while maintaining its high affinity for hDAT (human dopamine transporter) and simultaneously achieving adequate lipophilicity. One of the candidates, [68Ga]Ga‐HBED‐hexadiyne‐tropane, showed an IC50 value of 66 nM, together with a log D 7.4 of 0.96. A μPET study in a hemi‐parkinsonian rat model showed a fast wash‐out of the tracer, and no specific uptake in the brain, thus implying an inability to penetrate the BBB., Tracer metal: Tropanes are commonly used in radiopharmacy to assess the status of the dopaminergic system. However, no suitable radiometal‐labelled tracers are available for PET, primarily owing to the challenge of crossing the blood–brain barrier and loss of affinity through chelator attachment. We have developed 68Ga‐labelled phenyltropanes showing promising affinity and lipophilicity, and conducted a first PET study.

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

Species of Hyoscyamus are rich sources of medicinally important tropane alkaloids, which have anticholinergic, antispasmodic and sedative effects and are competitive inhibitors of acetylcholine. The application of nanotechnology and nanomaterials for elicitation is rapidly expanding and recent research indicates that silicon dioxide nanoparticles (SiO2 NPs) can be used as an efficient elicitor to increase the production of hyoscyamine and scopolamine in Hyoscyamus species. Thus, in this work, the effect of SiO2 NPs (0, 25, 50, 100 and 200 mg L−1) with two treatment times (24 and 48 h) on the growth rate, total phenol and flavonoid content (TPC, TFC), antioxidant enzyme activity, tropane alkaloid yield and pmt (putrescine N-methyltransferase) and h6h (hyoscyamine 6 -hydroxylase) gene expression levels in hairy roots of two Hyoscyamus species (H. reticulatus and H. pusillus) was investigated. The highest TPC and TFC accumulation was obtained in H. reticulatus elicited by SiO2 NPs (100 and 200 mg L−1), respectively, at 24 h of treatment. High-performance liquid chromatography (HPLC) revealed the highest amount of hyoscyamine (140.15 μg g−1 FW) and scopolamine (67.71 μg g−1 FW) accumulated in H. reticulatus transformed roots treated with 100 mg L−1 SiO2 NPs at 24 h, with a respective increase of 1212% and 272% compared to non-treated roots. In H. pusillus, the highest hyoscyamine (7.42 μg g−1 FW) and scopolamine (15.56 μg g−1 FW) production (about 82% and 241% higher, respectively, compared to the lowest amounts) was achieved with 25 and 100 mg L−1 SiO2 NPs, respectively, at 48 h of treatment. Semi-quantitative RT-PCR analysis determined the highest expression level of pmt and h6h genes in H. reticulatus transformed roots supplemented with 100 mg L−1 SiO2 NPs.

This Short Review highlights various synthetic approaches to bridged azabicyclic ring systems containing seven-membered carbocyclic rings. Such ring systems are common to a number of biologically active natural products. The seven-membered ring in such systems is generally formed in one of four ways: 1) cyclization of an acyclic precursor; 2) ring expansion or rearrangement of a different ring size; 3) cycloaddition; and 4) use of a synthetic building block with the seven-membered ring already present. Representative examples of each approach from both total synthesis and methodological studies are discussed, with an emphasis on work publishedin the last twenty years.1 Introduction2 Cyclization Reactions3 Ring Expansions and Rearrangements4 Cycloadditions5 Strategies Involving Seven-Membered Ring Building Blocks6 Conclusion

Missense mutations that give rise to protein misfolding are rare, but collectively, defective protein folding diseases are consequential. Folding deficiencies are amenable to pharmacological correction (pharmacochaperoning), but the underlying mechanisms remain enigmatic. Ibogaine and its active metabolite noribogaine correct folding defects in the dopamine transporter (DAT), but they rescue only a very limited number of folding-deficient DAT mutant proteins, which give rise to infantile Parkinsonism and dystonia. Herein, a series of analogs was generated by reconfiguring the complex ibogaine ring system and exploring the structural requirements for binding to wild-type transporters, as well as for rescuing two equivalent synthetic folding-deficient mutants, SERT-PG601,602AA and DAT-PG584,585AA. The most active tropane-based analog (9b) was also an effective pharmacochaperone in vivo in Drosophila harboring the DAT-PG584,585AA mutation and rescued 6 out of 13 disease-associated human DAT mutant proteins in vitro. Hence, a novel lead pharmacochaperone has been identified that demonstrates medication development potential for patients harboring DAT mutations.

The synthesis of tropanes via a microwave‐assisted, stereoselective 6π‐electrocyclic ring‐opening/ Huisgen [3+2]‐cycloaddition cascade of cyclopropanated pyrrole and furan derivatives with electron‐deficient dipolarophiles is demonstrated. Starting from furans or pyrroles, 8‐aza‐ and 8‐oxabicyclo[3.2.1]octanes are accessible in two steps in dia‐ and enantioselective pure form, being versatile building blocks for the synthesis of pharmaceutically relevant targets, especially for new cocaine analogues bearing various substituents at the C‐6/C‐7 positions of the tropane ring system. Moreover, the 2‐azabicyclo[2.2.2]octane core (isoquinuclidines), being prominently represented in many natural and pharmaceutical products, is accessible via this approach., Cyclopropanated furans and pyrroles can be converted into tropanes and isoquinuclidines, featuring a 6π‐electrocyclic ring‐opening/ Huisgen [3+2]‐cycloaddition cascade of 2‐oxo‐ and 2‐aza‐bicyclo[2.1.0]‐hex‐3‐enes.

Tropane alkaloids are toxic secondary metabolites produced by a wide variety of plants that can be present in edible materials or animal feed. Several human poisoning cases through consumption of cereals were reported over the last years and highlighted the need for reliable and robust analytical methodologies for safety control. To rationalize analyses in high-throughput laboratory environments dealing with shorter and shorter turn-around-around time, the scope of our multi mycotoxins method was extended to the analysis of two regulated tropane alkaloids, namely atropine and scopolamine. Extraction procedure is based on the QuEChERS (Quick, Easy, Cheap, Efficient, Rugged, and Safe) approach followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) detection. Quantification is performed by the isotopic dilution approach using labelled isotopomers as internal standard. The procedure was validated at two fortification levels (0.5 µg/kg and 10 µg/kg) on different cereal-based products according to the European SANTE/12682/2019 document and performance parameters such as precision (RSD(r) ≤ 6%, RSD(iR) ≤ 6%) and recovery (82-114%) fulfilled its requirements. The limit of quantification (0.5 µg/kg) is low enough to ensure compliance with existing regulations. The method was further applied on 95 cereals and cereal-based products collected from Asian and African countries. All samples were found free of the two targeted TAs, with the exception of a rice-based product in which both atropine and scopolamine were quantified at 9.6 µg/kg and 2.6 µg/kg, respectively. A total of 29 cereals samples, shown to be free of both atropine and scopolamine were also analysed for mycotoxins. Aflatoxins, fumonisins, and deoxynivalenol were sporadically detected at levels below the maximum levels defined by the European Union legislation often considered as the most stringent regulation.

Setting of maximum limits for a number of plant alkaloids is under discussion in the EU. The novel method developed and optimized in this study enables simultaneous determination of 21 tropane alkaloids (TAs) and 33 pyrrolizidine (PAs) together with their N-oxides (PANOs). For analysis of aqueous-methanolic extract, reversed phase ultra-high-performance liquid chromatography and tandem mass spectrometry (RP-U-HPLC-MS/MS) was employed. The method was validated for frequently contaminated matrices (i) sorghum, (ii) oregano, and (iii) mixed herbal tea. The recoveries at two spiking levels were in the range of 82–115%, 80–106%, and 78–117%, respectively, and repeatabilities were less than 19% for all analyte/matrix combinations. As regards the achieved limits of quantification (LOQ), their values were in the range of 0.5–10 μg kg−1. The crucial problem encountered during method development, co-elution of multiple groups of isomeric alkaloids, was overcome by subsequent sample separation in the second chromatographic system, hydrophilic interaction liquid chromatography (HILIC), providing different separation selectivity. Lycopsamine, echinatine, and indicine (co-elution group 1) and N-oxides of indicine and intermedine (co-elution group 2), which could not be resolved on the commonly used RP column, were possible to separate fully by using the HILIC system.

A modified quick, easy, cheap, effective, rugged and safe method was established for simultaneous determination of atropine, anisodamine, and scopolamine in goji berries by using ultra-high-performance liquid chromatography with tandem mass spectrometry. The graphene/hexagonal boron nitride hybrids were prepared and first applied as a cleanup adsorbent. Compared to classical cleanup adsorbent (C18 ), the graphene/hexagonal boron nitride hybrids as adsorbent had better extraction efficiency for the detection of analytes. Under the optimal conditions, the proposed analytical method achieved satisfactory linearity (R2 > 0.995), and obtained desirable recoveries ranged from 77.4 to 94.0% with the relative standard deviation of 1.2-6.1% at the concentration levels of 3.2-13.4 µg/kg. The limits of quantitation of atropine, anisodamine, and scopolamine were, respectively, 3.2, 4.6, and 4.5 µg/kg with linearity ranged from 3.2 to 25.4 µg/kg. The modified quick, easy, cheap, effective, rugged, and safe sample preparation with ultra-high-performance liquid chromatography and tandem mass spectrometry method was successfully applied to evaluate the safety of goji berries collected from 30 plant areas in China, suggesting its applicability and suitability for the routine analysis of three tropane alkaloids in goji berries.

Atropa belladonna produces anticholinergic tropane alkaloids (TAs), such as hyoscyamine and scopolamine, which are synthesized in its lateral roots. Yet, the regulation of lateral root growth and TA biosynthesis remain poorly understood. In this study, an auxin-responsive transcription factor (AbLBD1), encoding a lateral organ boundaries domain protein (LBD), was functionally characterized from A. belladonna. Overexpression of AbLBD1 effectively promoted the initiation of lateral roots and adventitious roots of A. belladonna. The root biomass of AbLBD1-overexpressing lines was 1.48 to 2.45 fold higher that of the control. However, the contents of hyoscyamine and scopolamine were markedly reduced in AbLBD1-overexpressing lines. Gene expression analysis showed that the expression levels of TAs biosynthesis genes, namely AbPMT, AbPYKS, AbCYP82M3, and AbH6H, were greatly decreased in AbLBD1-overpressing lines. In conclusion, AbLBD1 plays a positive role in regulating lateral root development but a negative one in regulating TAs biosynthesis in A. belladonna. This study proved for the first time that AbLBD1 was a key auxin response regulator in root, which promotes initiation of lateral root and inhibits the biosynthesis of tropane alkaloids.

Atropa belladonna L. is an important medicinal plant due to the biosynthesis of hyoscyamine and scopolamine; two important tropane alkaloids, mostly well known for their anticholinergic properties acting on the parasympathetic nervous system. This study aimed to analyze the effect of methyl jasmonate (MJ), at different concentrations, on hyoscyamine and scopolamine production from in vitro hairy root culture and propagated plantlets of A. belladonna L. Gene expression analysis of two key enzymes PMT and H6H responsible for the biosynthesis of hyoscyamine and scopolamine, respectively, was also carried out under MJ application. MJ decreased the growth parameters in both plantlets and hairy root cultures. In shoots of the plantlets, MJ increased significantly the hyoscyamine and scopolamine contents to the highest value of 3.32 mg g−1 DW and 0.5 mg g−1 DW, respectively. Moreover, MJ up-regulated pmt2 and h6h gene expression in root of the plantlets, which is the site of biosynthesis of hyoscyamine and scopolamine, while the accumulation occurs in the aerial parts. In hairy root culture, the hyoscyamine content increased as the MJ concentration increased, and reached its highest level of 2.1 mg g−1 DW at 0.04 mM MJ, showing the same pattern as for pmt2 gene expression. Nevertheless, the scopolamine content decreased by increasing the MJ concentration as it also showed a reverse pattern compared to the h6h gene expression, presumably due to the negative effect of hyoscyamine accumulation on H6H enzyme activity.

Recent research progress on the “second generation” type III polyketide synthases is summarized. This class of enzymes catalyzes unusual condensation chemistries of CoA thioesters to generate various core structures of medicinally important plant secondary metabolites, including the R1–C–R2 scaffold of alkyl quinolones, curcuminoids, as well as the 8-azabicyclo[3.2.1]octane ring of tropane alkaloids. The discovery of this fascinating enzyme superfamily provides excellent opportunities for the manipulation of the enzyme reactions to expand the supply of natural and unnatural molecules for future drug development. Electronic supplementary material The online version of this article (10.1007/s11418-020-01414-9) contains supplementary material, which is available to authorized users.

Background:Tropane Alkaloids (TAs) are important drugs for curing many diseases in the medical industry.Methods:To sustainably exploit TA resources in endangered traditional Tibetan herbs, the hairy root (HR) systems of Przewalskia tangutica Maxim. and Anisodus tanguticus Maxim. were compared under the same culture conditions.Results:The results indicated that both the Agrobacterium rhizogenes strains and explants affected the HR induction frequency, MSU440, A4 and LBA9402 strains could induce hairy roots following infection of cotyledon and hypocotyl of A. tanguticus while LBA9402 could not induce HR on either explants of P. tangutica. The efficiency of LBA9402 was higher than A4 and MSU440 on A. tanguticus and A4 was better strain than MSU440 on P. tangutica. The hypocotyl explant was more suitable for P.tangutica and cotyledon explant was better for A.tangutica with a transformation frequency of 33.3% (P. tangutica) and 82.5% (A. tanguticus), respectively. In a flask reactor system, both the growth curves of HR for two species both appeared to be “S” curve; however, the HR of P. tangutica grew more rapidly than that of A. tanguticus, and the latter accumulated more biomass than the former. As the culture volume increased, the HR proliferation coefficient of both the species increased. HPLC analysis results showed that the content of TAs in the HR of P. tangutica was 257.24mg/100g·DW, which was more than that of A. tanguticus HR (251.08mg/100g·DW), and the anisodamine in the Pt- HR was significantly higher than that in At-HR. Moreover, tropane alkaloids in the HR of the two species were all significantly higher than that of the roots of aseptic seedlings.Conclusion:Our results suggest that HR of P. tangutica and A. tanguticus both could provide a useful platform for sustainable utilization of two Tibetan medicinal plants in the Qinghai-Tibetan Plateau in the future.

Structurally fused heterocycles encompassing a centerpiece of benzotropane are significant scaffolds with a plethora of promising biological activities, but such molecular architectures pose a long-standing daunting synthetic challenge. Herein, we reported a highly efficient asymmetric cyclodearomatization of 2-nitrobenzofurans with cyclic azomethine ylides by employing bifunctional phosphonium salts as phase-transfer catalysts. Under optimized reaction conditions, a diverse array of polycyclic benzofused tropane derivatives with four contiguous 4°/3° stereocenters were readily synthesized in both high yields and diastereoselectivities with up to >99% ee. The practicality and utility of this protocol were further demonstrated by the scaled-up reaction and facile elaborations. Moreover, preliminary investigations into their antitumor activities were also presented.

A simple but effective method was developed for the ultra-trace determination of two tropane alkaloid residues, hyoscyamine and scopolamine, in honey. Honey samples were dissolved in a concentrated aqueous solution of sodium acetate from which the alkaloids were subsequently extracted using acetonitrile. The resulting extract was analyzed using HILIC–ESI-MS/MS without any additional extract cleanup or enrichment steps. Enhanced sensitivity associated with the combination of HILIC and ESI-MS/MS techniques permitted the quantitation of the two tropane alkaloids at concentrations as low as 0.01 µg kg−1. The use of isotopically labelled analogs permitted accuracies ranging from 86.9 to 106.1% for honey samples fortified at 0.02, 0.2 and 2 µg kg−1 with precisions ranging from 0.8 to 6.2%. The estimated method detection limits for hyoscyamine and scopolamine were 0.002 and 0.003 µg kg−1, respectively. Twenty-three samples of honey were analyzed and only two samples were found to contain residues of the tropane alkaloids at concentrations above the limit of quantitation of 0.01 µg kg−1. Hyoscyamine was detected at 0.012 µg kg−1 in two honey samples, one of which also contained 0.012 µg kg−1 of scopolamine.

Tropanes and related bicyclic alkaloids are highly attractive compounds possessing a broad biological activity. Here we report a mild and simple protocol for the synthesis of N-arylated 8-azabicyclo[3.2.1]octane and 9-azabicyclo[3.3.1]nonane derivatives. It provides these valuable bicyclic alkaloid skeletons in good yields and high levels of diastereoselectivity from simple and readily available starting materials using visible-light photoredox catalysis. These bicyclic aniline derivatives are hardly accessible via the classical Robinson tropane synthesis and represent a particularly attractive scaffold for medicinal chemistry. This unprecedented annulation process takes advantage of the unique reactivity of ethyl 2-(acetoxymethyl)acrylate as a 1,3-bis radical acceptor and of cyclic N,N-dialkylanilines as radical 1,3-bis radical donors. The success of this process relies on efficient electron transfer processes and highly selective deprotonation of aminium radical cations leading to the key α-amino radical intermediates.

Methyl 4-(1-methylpyrrolidin-2-yl)-3-oxobutanoate and hygrine are important biosynthetic intermediates for tropane alkaloids. We have developed a concise method to synthesize these two compounds from the key intermediate N-methylpyrrolinium cation. Methyl 4-(1-methylpyrrolidin-2-yl)-3-oxobutanoate and hygrine were obtained in four and six steps from commercially available 4,4-diethoxybutylamine with overall yields of 42% and 25%, respectively.

Ornithine decarboxylase (ODC) plays an important role in various biological processes; however, its role in plant secondary metabolism, especially in the biosynthesis of tropane alkaloids (TAs) suc...

Conformationally restrained exo-isomers of N-Boc-protected spiro[2′,2′-difluorocyclopropane-3′,2′-tropanes] were synthesized in 62–83% yield via absolutely diastereoselective cycloaddition of CF2, generated in situ from Me3SiCF3/NaI in refluxing THF, to double bonds of 3-alkylidenetropanes. Standard removal of Boc protecting groups afforded corresponding exo-spiro[2′,2′-difluorocyclopropane-3′,2′-tropane] hydrochlorides in 82–94% yields. DFT and CCSD(T) calculations revealed that the observed exo-selectivity of difluorocarbene addition is likely to be caused by a lower activation barrier of the exo-difluorocyclopropanation compared to the endo-reaction.

Identification and quantification of different metabolites under stress, especially protein, is a vital way to understand plant adaptation mechanism. We established an efficient protein extraction method from the tiny amount (100 mg) of root tips of non-model medicinal plant Hyoscyamus albus, using bead-beating cell disruption, TRIzol extraction, and sequential chemical protein solubilization. H. albus is very well known for biosynthesized of different secondary metabolites like hyoscyamine, tropane alkaloids and scopolamine. Our method is rational for sample preparation even in small-scale proteomics of recalcitrant tissue and allows proficient, reproducible and impurity-free protein extraction. This method allows high-quality 2DE in mini-gel format (25 µg of protein/gel) for hydrophilic and hydrophobic sub-proteomes and is compatible to high-sensitive matrix-assisted laser/desorption ionization quadrupole ion trap time-of-flight (MALDI-QIT-TOF) mass spectrometry (MS). A protocol using TRIzol is more effective and reproducible to sequential chemical extraction of both hydrophilic and hydrophobic membrane proteins. We also demonstrated cell disrupted together with dithiothreitol (DTT) and polyvinylpolypyrrolidone (PVPP) is more useful to prevent polymerization of the phenolic compound than commonly used added DTT and PVPP with TRIzol reagent. Despite the unavailability of genomic sequence database, the efficacy of the protocol was also confirmed by MS/MS ion searches. J Bangladesh Agril Univ 17(4): 430–436, 2019

Structures of alkaloid convolvine (1) isolated from Convolvulus subhirsutus and its derivatives-convolamine(N-methylconvolvine) (2) and hydrochloride of N-benzylconvolvine (3) have been determined by single crystal X-ray diffraction technique. Compounds were crystallized in monoclinic space groups having four molecules in unit cell. All compounds contain a bicyclic ring system of tropane, where piperidine rings in all case adopt chair conformation. Hydrogen atom and methyl- and benzyl-substituents located in nitrogen atom of studied compounds occupy equatorial positions. The substituent of tropane core- the veratroyloxy group containing in all compound molecules is an α-axial oriented relative to the tropane core. In crystal structures of compound 1 and 2, the molecules are located in the distance of van der Waals interactions. The H-bond between the anion Cl and the proton of the N atom is observed in the crystal of N-benzylconvolvine hydrochloride (Cl···N 3.337 Å, Cl···H 2.42 Å and Cl-H-N 175°).

A straight synthetic approach to fused polycyclic tropane scaffold formation through an asymmetric dearomatization cycloaddition process of 2-nitrobenzofurans with cyclic azomethine ylides was successfully developed. In the presence of a chiral copper complex, derived from Cu(OAc)

It is thought that natural selection exerted by herbivores on plants has promoted the evolution of plant traits that function as defence. However, such selective pressures may vary spatially differentiating populations in plant defence phenotypes. Yet, to ascertain the role of natural selection on phenotypic differentiation between populations, it is necessary to discard other evolutionary processes like genetic drift. Evolutionary biologists have designed approaches to determine whether population differentiation has been produced by natural selection in contrast to random processes as a null hypothesis. To accomplish this, we compare the magnitude of differentiation among populations in plant defence against herbivores (selection) and in neutral loci (genetic drift). Our study system is the plant Datura stramonium, whose anti-herbivore defence includes tropane alkaloids and foliar trichomes, and its specialized herbivorous insects. We selected two geographically close natural populations of D. stramonium in Central Mexico and estimated, under controlled conditions, population differentiation at neutral loci (microsatellites) and defence traits (concentration of tropane alkaloids and leaf trichome density). Results indicate very low genetic differentiation at neutral loci between populations but strong and significant phenotypic differentiation in putative defence traits. The average values of tropane alkaloids and leaf trichome density were higher in Ticuman than in Teotihuacan. Twelve out of 21 individual tropane alkaloids were significantly more abundant in plants from Ticuman, and the relative proportion of three of them contrasted markedly. Thus, results point that differentiation between populations of D. stramonium results from natural selection on defence traits.

Some medicinal plants of the Solanaceae produce pharmaceutical tropane alkaloids (TAs), such as hyoscyamine and scopolamine. Littorine is a key biosynthetic intermediate in the hyoscyamine and scopolamine biosynthetic pathways. However, the mechanism underlying littorine formation from the precursors phenyllactate and tropine is not completely understood. Here, we report the elucidation of littorine biosynthesis through a functional genomics approach and functional identification of two novel biosynthesis genes that encode phenyllactate UDP-glycosyltransferase (UGT1) and littorine synthase (LS). UGT1 and LS are highly and specifically expressed in Atropa belladonna secondary roots. Suppression of either UGT1 or LS disrupted the biosynthesis of littorine and its TA derivatives (hyoscyamine and scopolamine). Purified His-tagged UGT1 catalysed phenyllactate glycosylation to form phenyllactylglucose. UGT1 and LS co-expression in tobacco leaves led to littorine synthesis if tropine and phenyllactate were added. This identification of UGT1 and LS provides the missing link in littorine biosynthesis. The results pave the way for producing hyoscyamine and scopolamine for medical use by metabolic engineering or synthetic biology.

A sensitive UHPLC-ESI-MS/MS method, coupled with a suitable extraction protocol, was developed and validated for the rapid detection of four tropane alkaloids (anisodamine, atropine, homatropine, scopolamine) in herbal teas and herbal extracts. The method offers good sensitivity, accuracy and precision. The protocol is suitable for routine determination of tropane alkaloids in dried plants and extracts sold as food supplements or phytotherapics, and was applied to 60 commercial samples. Anisodamine, atropine and/or scopolamine were found in 4 herbal teas samples (LOQ–69 μg/kg for atropine and 50 μg/kg for scopolamine) and in a Lepidium meyenii extract sold as food supplement (traces of anisodamine). The level of contamination do not to pose a direct risk for the consumers if compared to the acute reference dose indicated by EFSA, but the detection confirms the need for suitable methods and careful screenings in order to determine a more precise evaluation of dietary intake for tropane alkaloids.

Recently, surface-enhanced Raman Spectroscopy (SERS) has been widely applied for rapid detection of trace targets in various fields. However, two key issues are still being explored: (1) how to for...

Atropabelladonna sistematik olarak Solanaceae ailesinde yer alan çok yıllık otsu birbitkidir. Çiçekleri yeşilimsi mor, yaprakları oval, meyveleri siyah renkte veparlaktır. Güzel avrat otu; Atropin, Skololamin, Belladonnin, Hyosiyamin,Apoatropin gibi önemli alkoloidleri kapsar. En önemli kimyasal maddesi iseAtropin’ dir. İçerdiği etken madde 1809 yılında yalıtılmış ve 1819 yılındansonrada alkaloid olarak sınıflandırılmıştır. En yüksek alkaloidoranı yeşil yapraklarında ve meyvelerinde olmasına rağmen bitkinin bütünkısımları alkaloid içerir. Birçok kodekstealkaloid oranının % 3’ün altında olmaması istenir. Güzel avratotu (Atropa), kapsadığı etkenmaddelerden dolayı eskiden bu yana birçok ülkede ekilen bir bitkidir, esasalkoloid olan atropinin sentetik olarak elde edilen türevleri eczacılıktaönemli bir yere sahiptir. Ülkemizde de Atropa belladonna sahip olduğu tropanalkaloidleri ile bitkisel kökenli ilaçlar arasında önemli bir yere sahiptir., Atropabelladonna is a perennial herbaceous plant that is systenatically found in Solanaceaefamily. The flowers are greenish purple, the leaves are oval, the fruits areblack and bright. Deadly nightshade includes important alkaloids suvh asatropine, skololamine, belladonnaine, hypocaine,apoatropine. The most importantchemical substance is atropine. İts active ingredient was isolated in 1809 andlater classified as alkaloid by 1819. All parts of the plantcontain alkaloids although the highest alkaloid ratio is in the green leavesand in the fruites. İt is the desirable that the ratio of several cotechealkaloids is not less than 3 %. Deadly Nightshade is a plantthat has been planted in many countries since ancient times due to ıts activeingredients and the synthetically derived derivatives of atropine whis is themain alkaloid have important place in pharmacy. İn our country Atropa belladonna has an important placeamong tropane alkaloids and herbal medicines.

Solanaceae plant seeds, which contain high concentrations of tropane alkaloids, have not been studied in real conditions of proofing and baking processes. In this work both lab vial trials and buckwheat and millet flour samples, contaminated with two species of Solanaceae plants, Datura stramonium and Brugmansia arborea, were undergone to proofing (37 °C) and baking (190 °C) processes. For the determination of tropane alkaloids, a simple solid-liquid extraction with methanol:water 2:1 (v/v) containing 0.5% acetic acid was used to extract the targeted compounds, whereas a chromatographic method employing a Zorbax C18 column coupled to an Exactive-Orbitrap analyser was used for their determination. The results indicate that concentrations of tropane alkaloids decrease under proofing conditions (degradation between 13 and 95%), while they are almost disappeared under baking conditions (degradation between 94 and 100%). Some degradation pathways have been clarified, showing that most of the compounds degrade into tropane and tropine, and into tropine and tropinone under proofing and baking conditions respectively.

In this study, the degradation of tropane alkaloids in pasta under boiling (100 °C during 10 min) and tea making (100 °C and let cool 5 min) conditions has been evaluated for the first time. Pasta and green tea were contaminated with Datura Stramonium and Brugmansia Arborea seeds (pasta and green tea), whereas coca leaf tea was directly analysed. The compounds were extracted using solid-liquid extraction coupled to a preconcentration stage (only for the cooking water), and the compounds were analysed by liquid chromatography coupled to mass spectrometry (Exactive-Orbitrap analyser). Degradation studies indicate that concentration of tropane alkaloids decreases, and it depends on the compound, observing the highest degradation for tropinone, tropane, cuscohygrine and tropine, as well as it was observed that compounds migrated to the aqueous phase during cooking step. Finally, post-targeted analysis was performed and other tropane alkaloids were found, as scopine, tigloidine or convolvine, showing a similar behaviour under cooking conditions.

Przewalskia tangutica is a traditional medicinal plant from Tibet used for the analgesic effect from the tropane alkaloids (TAs) produced by the plant. Its roots have the highest yield of hyoscyamine in all plant species and so have been overharvested becoming an endangered medicinal plant species. Metabolic engineering is a good way to improve the yield of TAs in plants. In our study, two functionally distinct tropinone reductases genes, PtTRI and PtTRII, were cloned from P. tangutica and the functional divergence were characterized. The enzyme kinetics of PtTRI and PtTRII were investigated. The phylogenetic analysis classified them into different clades: PtTRI and PtTRII were in the clade of tropine-forming reductase and pseudotropine-forming reductase, respectively. We found PtTRI to be expressed in the roots but less in leaves, whereas PtTRII was expressed in the roots at higher levels than in the leaves. The kinetic parameters (Km , Vmax , and Kcat ) were analyzed using purified recombinant enzymes at their optimum pH. Enzymatic analysis results showed that tropinone is a better substrate for PtTRII compared with PtTRI, suggesting that PtTRII might be a potential gene target for TA biosynthesis engineering. Compared with the reported TRIs, PtTRI exhibited a higher affinity for tropinone.