Tropine

Detection and identification of ACP-105 and its metabolites in equine urine using LC/MS/MS after oral administration.[Pubmed:32852865]

Drug Test Anal. 2021 Feb;13(2):299-317.

ACP-105 is a novel nonsteroidal selective androgen receptor modulator (SARM) with a tissue-specific agonist effect and does not have side effects associated with the use of common androgens. This research reports a comprehensive study for the detection of ACP-105 and its metabolites in racehorses after oral administration (in vivo) and postulating its structures using mass spectrometric techniques. To obtain the metabolic profile of ACP-105, a selective and reliable LC-MS/MS method was developed. The chemical structures of the metabolites were determined based on their fragmentation pattern, accurate mass, and retention time. Under the current experimental condition, a total of 19 metabolites were detected in ACP-105 drug administered equine urine samples. The study results suggest the following: (1) ACP-105 is prone to oxidation, which gives corresponding monohydroxylated, dihydroxylated, and trihydroxylated metabolites; (2) along with oxidation, there is a possibility of elimination of water molecule (dehydration) from the third position of the Tropine moiety, resulting in the dehydrated analogs of corresponding monohydroxylated, dihydroxylated, and trihydroxylated metabolites; (3) from the study on the metabolites using LC-MS/MS, it is clear that the fragmentation pattern is identical and a great number of fragment ions are common in all the metabolites and the parent drug. (4) The ACP-105 and its metabolites were detected for up to 72 h; thus, the result is a valuable tool for evaluating its use and/or misuse in sport.

Dispersive solid-phase extraction of racemic drugs using chiral ionic liquid-metal-organic framework composite sorbent.[Pubmed:32823100]

J Chromatogr A. 2020 Sep 13;1627:461395.

Nowadays, enantioseparation of racemic pharmaceuticals in preparations is a prime concern by drug authorities across the globe. In the present work, it was attempted to develop novel enantioselective extraction method for five clinically used drugs (atenolol, propranolol, metoprolol, racecadotril, and raceanisodamine in their tablets) as racemates. The enantioselective solid-liquid extraction of these racemic drugs was carried out successfully by the use of chiral ionic liquid (CIL) in combination with a metal organic framework (MOF) for the first time. The composite CIL@MOF was synthesized from Tropine based chiral ionic liquids with L-proline anion ([CnTr][L-Pro], n=3-6) and HKUST-1 type MOF, which was comprehensively characterized before being used as sorbent for enantioselective dispersive solid-liquid extraction. Preliminary selection of appropriate CIL was carried out on thin layer chromatography (TLC); under the joint participation of copper ion in the developing reagent, [C3Tr][L-Pro] ionic liquid showed better resolution performance with DeltaRf value of 0.35 between the enantiomers was obtained for racemic atenolol. Moreover, the effect of copper salt dosage, amount of CIL, soli-liquid ratio and extraction time were investigated. The optimal conditions were obtained after thorough investigations; i.e. sample solution: ethanol, elution solvent: methanol, solid-liquid ratio: 12.5 mg:50 mL, amount of copper salt: 8 mg L(-1), amount of impregnated CIL: 30% and extraction time of 30 min. As a result, enantiomeric excess values are 90.4%, 95%, 92%, 81.6% and 83.2% for atenolol, propranolol, metoprolol, racecadotril and raceanisodamine, respectively. The developed enantioselective method was validated following ICH guidelines and it was proved to be simple, effective and enantioselective way for separation of racemic pharmaceuticals with similar behaviors.

Functional genomics analysis reveals two novel genes required for littorine biosynthesis.[Pubmed:31705812]

New Phytol. 2020 Mar;225(5):1906-1914.

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.

Preconcentration of tropane alkaloids by a metal organic framework (MOF)-immobilized ionic liquid with the same nucleus for their quantitation in Huashanshen tablets.[Pubmed:31637371]

Analyst. 2019 Nov 18;144(23):6989-7000.

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 mug L-1 with a linear range from 100-500 mug 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.

Engineering a microbial biosynthesis platform for de novo production of tropane alkaloids.[Pubmed:31406117]

Nat Commun. 2019 Aug 12;10(1):3634.

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.

Ultrasonic Extraction of Tropane Alkaloids from Radix physochlainae Using as Extractant an Ionic Liquid with Similar Structure.[Pubmed:31404959]

Molecules. 2019 Aug 9;24(16). pii: molecules24162897.

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.

Degradation of tropane alkaloids in baked bread samples contaminated with Solanaceae seeds.[Pubmed:31229117]

Food Res Int. 2019 Aug;122:585-592.

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 degrees C) and baking (190 degrees 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.

De Novo Production of the Plant-Derived Tropine and Pseudotropine in Yeast.[Pubmed:31181154]

ACS Synth Biol. 2019 Jun 21;8(6):1257-1262.

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.

Biochemical characterization reveals the functional divergence of two tropinone reductases from Przewalskia tangutica.[Pubmed:31051047]

Biotechnol Appl Biochem. 2019 Jul;66(4):597-606.

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.

A Nickel(II) Nitrite Based Molecular Perovskite Ferroelectric.[Pubmed:31050113]

Angew Chem Int Ed Engl. 2019 Jun 24;58(26):8857-8861.

The X-site ion in organic-inorganic hybrid ABX3 perovskites (OHPs) varies from halide ion to bridging linkers like HCOO(-) , N3 (-) , NO2 (-) , and CN(-) . However, no nitrite-based OHP ferroelectrics have been reported so far. Now, based on non-ferroelectric [(CH3 )4 N][Ni(NO2 )3 ], through the combined methodologies of quasi-spherical shape, hydrogen bonding functionality, and H/F substitution, we have successfully synthesized an OHP ferroelectric, [FMeTP][Ni(NO2 )3 ] (FMeTP=N-fluoromethyl Tropine). As an unprecedented nitrite-based OHP ferroelectric, the well-designed [FMeTP][Ni(NO2 )3 ] undergoes the ferroelectric phase transition at 400 K with an Aizu notation of 6/mmmFm, showing multiaxial ferroelectric characteristics. This work is a great step towards not only enriching the molecular ferroelectric families but also accelerating the potential practical applications.

Effect of tea making and boiling processes on the degradation of tropane alkaloids in tea and pasta samples contaminated with Solanaceae seeds and coca leaf.[Pubmed:30857698]

Food Chem. 2019 Jul 30;287:265-272.

In this study, the degradation of tropane alkaloids in pasta under boiling (100 degrees C during 10min) and tea making (100 degrees C and let cool 5min) 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.

Extraction and quantitative analysis of tropane alkaloids in Radix physochlainae by emulsion liquid membrane with tropine-based ionic liquid.[Pubmed:30429086]

J Chromatogr A. 2019 Jan 4;1583:9-18.

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.