Assessment of tropane alkaloid levels in Brazilian buckwheat flour products: A novel LC-UHPLC-MS/MS approach using solid-liquid extraction at low temperature.

In recent years, the monitoring of tropane alkaloids, specifically hyoscyamine and scopolamine, in food has become a pressing concern. This is due to increasing reports of food contamination with these compounds worldwide, raising awareness about the potential risks associated with their consumption. A novel method is proposed here for the determination of the sum of (+)-hyoscyamine, (-)-hyoscyamine, and (-)-scopolamine in buckwheat-based matrices, using solid-liquid extraction at low temperature and quantification by bidimensional chromatography coupled to tandem mass spectrometry. The validated method presented a linear response in the concentration range of 2.5-15 µg kg-1 (r > 0.99). The precision and accuracy were in the ranges from 0.8 to 11.0 % and from 96 to 103 %, respectively. The limit of quantification (LOQ) was 2.5 µg kg-1. No contamination was found at levels above the LOQ in any of the 18 samples analyzed (buckwheat flour, grains, and gluten-free mix).

Evaluation of Tropane Alkaloids in Teas and Herbal Infusions: Effect of Brewing Time and Temperature on Atropine and Scopolamine Content.

Atropine and scopolamine belong to the tropane alkaloid (TA) family of natural toxins. They can contaminate teas and herbal teas and appear in infusions. Therefore, this study focused on analyzing atropine and scopolamine in 33 samples of tea and herbal tea infusions purchased in Spain and Portugal to determine the presence of these compounds in infusions brewed at 97 °C for 5 min. A rapid microextraction technique (µSPEed®) followed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was used to analyze the selected TAs. The results showed that 64% of the analyzed samples were contaminated by one or both toxins. White and green teas were generally more contaminated than black and other herbal teas. Of the 21 contaminated samples, 15 had concentrations above the maximum limit for liquid herbal infusions (0.2 ng/mL) set by Commission Regulation (EU) 2021/1408. In addition, the effects of heating conditions (time and temperature) on atropine and scopolamine standards and naturally contaminated samples of white, green, and black teas were evaluated. The results showed that at the concentrations studied (0.2 and 4 ng/mL), there was no degradation in the standard solutions. Brewing with boiling water (decoction) for 5 and 10 min allowed for higher extraction of TAs from dry tea to infusion water.

Rapid Detection and Determination of Scopolamine in the Leaf Extract of Black Henbane (Hyoscyamus niger L.) Plants Using a Novel Nanosensor.

BACKGROUND: Scopolamine is among the most essential tropane alkaloids used to remedy various nervous system disorders such as urinary incontinence, motion sickness, and spasmodic movements because of its anticholinergic and antispasmodic effects. OBJECTIVE: In this study, an optical nanosensor was fabricated using nano-Dragendorff's reagent to detect and determine scopolamine in different plant parts at different stages of growth. METHOD: For fabrication of the sensing phase, GO-g-PCA/DR was synthesized by encapsulation of Dragendorff's reagent (DR) on the graphene oxide grafted with poly citric acid (GO-g-PCA) with ultrasonication for 15 min and stirred for 80 min at room temperature, and then it was immobilized on a triacetyl cellulose membrane. The kinetic absorption profiles were recorded at 360 nm, which is concerned with the reaction between immobilized GO-g-PCA/DR and different concentrations of scopolamine. RESULTS: The nanosensor showed a rapid, strong, and stable response to the scopolamine solution with changing the absorption spectrum at 360 nm. The reaction was completed in a period of 300 s. The SEM, AFM, and FT-IR analysis of nanocomposites and nanosensors show the successful synthesis of GO-g-PCA/DR and the reaction between nanosensor and scopolamine. All experiments were performed at the wavelength of 360 nm, room temperature, pH 7 (the scopolamine solution pH), and 300 s. The nanosensor had a linear range of 0.65 to 19.63 µg/mL and 0.19 ± 0.025 µg/mL as the limit of detection for scopolamine determination. In order to reuse the designed nanosensor, it was recovered with ethanol, and the color ultimately returned to its original state. CONCLUSIONS: This in situ nanosensor can determine the scopolamine in real samples with easy reversibility, extended lifetime, and reproducibility of the sensing phase response. HIGHLIGHTS: A sensitive, precise, and fast response optical nanosensor is designed for in situ determination of scopolamine in real samples.

Neuroprotective Effect of Yucca schidigera Roezl ex Ortgies Bark Phenolic Fractions, Yuccaol B and Gloriosaol A on Scopolamine-Induced Memory Deficits in Zebrafish.

Y. schidigera contains a number of unusual polyphenols, derivatives of resveratrol and naringenin, called spiro-flavostilbenoids, which have potent in vitro anti-inflammatory, antioxidant, and moderate cholinesterase inhibitory activities. To date, these compounds have not been tested in vivo for the treatment of neurodegenerative diseases. The aim of the present study was to evaluate the effects of both single spiro-flavostilbenoids (yuccaol B and gloriosaol A) and phenolic fractions derived from Y. schidigera bark on scopolamine-induced anxiety and memory process deterioration using a Danio rerio model. Detailed phytochemical analysis of the studied fractions was carried out using different chromatographic techniques and Nuclear Magnetic Resonance (NMR). The novel tank diving test was used as a method to measure zebrafish anxiety, whereas spatial working memory function was assessed in Y-maze. In addition, acetylcholinesterase/butyrylcholinesterase (AChE/BChE) and 15-lipooxygenase (15-LOX) inhibition tests were performed in vitro. All pure compounds and fractions under study exerted anxiolytic and procognitive action. Moreover, strong anti-oxidant capacity was observed, whereas weak inhibition towards cholinesterases was found. Thus, we may conclude that the observed behavioral effects are complex and result rather from inhibition of oxidative stress processes and influence on cholinergic muscarinic receptors (both 15-LOX and scopolamine assays) than effects on cholinesterases. Y. schidigera is a source of substances with desirable properties in the prevention and treatment of neurodegenerative diseases.

Green extraction approach based on µSPEed® followed by HPLC-MS/MS for the determination of atropine and scopolamine in tea and herbal tea infusions.

A high throught methododology based on a green extraction technique, µSPEed®, followed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) has been proposed for the analysis of atropine and scopolamine in tea and herbal tea infusions. For this, a digiVOL® Digital Syringe was used with different sorbents and working conditions to obtain a fast and efficient µSPEed® extraction. The best performance was achieved with a PS/DVB sorbent phase, sample loading of 5 × 500 µL and elution with 2 × 100 µL aliquots of methanol. The strategy based on µSPEed® followed by HPLC-MS/MS was validated, attaining quantitation limits lower than 0.15 ng mL-1 and recoveries between 94 and 106% for both analytes and applied to seventeen tea and herbal tea infusions. Fourteen infusions showed contamination with one or both analytes above the maximum content legislated (sum of atropine and scopolamine < 0.2 ng mL-1).

Development of CE-C4D Method for Determination Tropane Alkaloids.

A fast method for the determination of tropane alkaloids, using a portable CE instrument with a capacitively coupled contactless conductivity detector (CE-C4D) was developed and validated for determination of atropine and scopolamine in seeds from Solanaceae family plants. Separation was obtained within 5 min, using an optimized background electrolyte consisting of 0.5 M acetic acid with 0.25% (w/v) ß-CD. The limit of detection and quantification was 0.5 µg/mL and 1.5 µg/mL, respectively, for both atropine and scopolamine. The developed method was validated with the following parameters-precision (CV): 1.07-2.08%, accuracy of the assay (recovery, RE): 101.0-102.7% and matrix effect (ME): 92.99-94.23%. Moreover, the optimized CE-C4D method was applied to the analysis of plant extracts and pharmaceuticals, proving its applicability and accuracy.

Determination of tropane alkaloids in cereals, tea and herbal infusions: Exploiting proficiency testing data as a basis to derive interlaboratory performance characteristics of an improved LC-MS/MS method.

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.

[Nitrogen metabolism and secondary metabolism regulation of Atropa belladonna by exogenous NO under NaCl stress].

Atropa belladonna seedlings were used as experimental materials and cultivated by soil culture method. Different concentrations(0,0.05,0.1,0.2,0.5 mmol·L~(-1))of NO donor sodium nitroprusside(SNP) were sprayed on the leaves. The effects of different concentrations of SNP and different treatment time(4,8,12,16 d) on nitrogen metabolism, secondary metabolite content, precursor content of tropane alkaloid synthesis pathway and expression of key enzyme genes under 100 mmol·L~(-1) NaCl stress were studied. The results showed that with the prolongation of salt stress, the nitrogen metabolism and the accumulation of secondary metabolites of A. belladonna were inhibited to some extent. After treatment with different concentrations of exogenous SNP, the ammonium nitrogen content decreased dramatically, and the contents of nitrate nitrogen, free amino acid, soluble protein and the activities of key enzymes of nitrogen metabolism(NR, GS, GDH) were all greatly improved; the contents of precursor amino acids(ornithine, arginine) and polyamines(Put, Spd, Spm) in the secondary metabolic pathway have increased to varying degrees. The qRT-PCR analysis showed that exogenous SNP treatment can effectively promote the high expression of key enzyme genes PMT, TRⅠ and H6H in the secondary metabolic pathway of A. belladonna, and the production of hyoscyamine and scopolamine were increased notably. In summary, the application of appropriate concentration of SNP can effectively alleviate the inhibition of salt stress on the nitrogen metabolism and secondary metabolism of Atropa belladonna, and enhance its salt tolerance. Overall, 0.1 mmol·L~(-1) and 0.2 mmol·L~(-1) SNP treatment achieved the most remarkable effect.

Simple interface for scanning chemical compounds on developed thin layer chromatography plates using electrospray ionization mass spectrometry.

A simple and cheap design for interfacing thin layer chromatography (TLC) with electrospray ionization mass spectrometry (ESI/MS) was developed to scan and characterize compounds on TLC plate. The developed TLC plate was rapidly and easily modified into two sawtooth-edged pieces that were positioned on an XYZ stage so that one of the triangular tips was pointed toward the MS inlet. A drop of methanol and high DC voltage was applied at the tip to induce ESI. After the analytes in the first tip were analyzed, the TLC piece was moved so that the second triangular tip was pointed toward the MS inlet for analysis. The process was repeated until all the triangular tips on the piece were analyzed. In this manner, the analytes, no matter visible or non-visible bands, were scanned and characterized. Since a 4.8 cm long TLC track were cut to 32 triangles on two sawtooth pieces for analysis, the spatial resolution of using the sawtooth TLC-ESI/MS for analysis is 1.5 mm/band. A mixture of dye standards and Datura metel flower extract was analyzed to demonstrate the capability of sawtooth TLC-ESI/MS on scanning and characterizing chemical compounds on the TLC plates. The limits of detection of the dye standards were between 0.25 and 2.5 ng/band. TLC bands containing alkaloids such as scopolamine and norscopolamine from the Datura metel flower extract were not visualized on the developed TLC track, but were successfully detected at different triangular tips using sawtooth TLC-ESI/MS. Based on these results, the Rf values of scopolamine and norscopolamine were determined.

Simultaneous quantification of atropine and scopolamine in infusions of herbal tea and Solanaceae plant material by matrix-assisted laser desorption/ionization time-of-flight (tandem) mass spectrometry.

RATIONALE: Atropine (Atr) and scopolamine (Scp) are toxic secondary plant metabolites of species within the Solanaceae genus that can accidentally or intentionally reach the food store chain by inaccurate harvesting of any plant material, e.g. for herbal tea infusions. Ingestion may cause severe anticholinergic poisoning thus requiring risk-oriented determination in food and beverages. The suitability of matrix-assisted laser desorption/ionization time-of-flight (tandem) mass spectrometry, MALDI-TOF MS(/MS), should be characterized for simultaneous analysis. METHODS: We herein present the first MALDI-TOF MS(/MS) procedure for quantitative determination of both alkaloids in herbal tea infusions and Solanaceae plant material. A standard additions procedure using triply deuterated Atr as internal standard was developed and validated. RESULTS: Tropane alkaloids were detected without interferences and the standard additions procedure allowed reliable quantification. Intraday and interday precision were less than 17% and corresponding accuracies were between 77% and 112%. Limits of detection in the spotting solution were found at 5 ng/mL (Atr) and 0.5 ng/mL (Scp). The assay was applied to diverse tea infusions as well as to berries and leaves of deadly nightshade and angel's trumpet. CONCLUSIONS: The usefulness of MALDI-TOF MS(/MS) for investigations of plant-derived samples to prove contaminations by small basic compounds was demonstrated. The elaborate procedure is reliable but quite laborious to obtain quantitative results, but MALDI-TOF MS(/MS) was also shown to be a valuable tool for rapid qualitative screening for Atr and Scp in plant extracts.

Screening of drugs and homeopathic products from Atropa belladonna seed extracts: Tropane alkaloids determination and untargeted analysis.

Homeopathic products are still a controversial issue in modern medicine, understood as complementary or alternative medicine (CAM). In this particular case, homeopathic products prepared from Atropa belladonna extracts may present specific problems due to the effects derived from its components. This article applies a simple, rapid, reliable method to the analysis of different homeopathic products obtained from Atropa belladonna; drugs containing high concentration of plant extracts; and Atropa belladonna seeds. The method was based on a simple solid-phase preconcentration method followed by ultra-high pressure liquid chromatography (UHPLC) coupled to high resolution mass spectrometry using Exactive-Orbitrap as an analyser. An in-house database was set and atropine and scopolamine were the compounds detected at highest concentrations in homeopathic products from Atropa belladonna extracts (4.57 and 2.56 µg/kg, respectively), in Belladonna ointment (4007 and 1139 µg/kg, respectively) and Belladonna seeds (338 and 32.1 mg/kg, respectively). Other tropane alkaloids such as tropine, apoatropine, aposcopolamine, tropinone, homatropine, and anisodamine were detected at lower concentrations (0.04-1.36 µg/kg). When untargeted analysis was performed, other tropane alkaloids were identified in the tested samples, such as ecgonine (0.003 µg/kg), benzoylecgonine (0.56 µg/kg), calystegines A (19.6 µg/kg), B (33.1 µg/kg), and C (1.01 µg/kg). Finally other compounds present in the homeopathic products, such as sugars (fructose, glucose, and lactose) or amino acids (valine, ornithine, leucine, and phenylalanine), were identified.

FRET-based nanobiosensor for detection of scopolamine in hairy root extraction of Atropa belladonna.

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.

Isolation of atropine and scopolamine from plant material using liquid-liquid extraction and EXtrelut® columns.

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.

Simultaneous determination of atropine and scopolamine in buckwheat and related products using modified QuEChERS and liquid chromatography tandem mass spectrometry.

A method was developed for the determination of atropine and scopolamine in buckwheat and related products. A modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) extraction procedure was evaluated. Dispersive solid phase extraction (d-SPE) was studied as clean-up step, using graphitized black carbon (GBC) and primary secondary amine (PSA). The extract was diluted with water (50:50, v/v) prior to chromatographic analysis. The method was validated and recoveries (except chia samples spiked at 10µg/kg) ranged from 75% to 92%. Intra and inter-day precision was lower than or equal to 17%. The limit of quantification of atropine and scopolamine was 0.4 and 2µg/kg, respectively. Eight types of samples (buckwheat, wheat, soy, buckwheat flour, buckwheat noodle, amaranth grain, chia seeds and peeled millet) were analyzed. Target compounds were not found above the detection limits of the method, but three transformation products of scopolamine (norscopine, hydroscopolamine and dihydroxyscopolamine) were putative identified in the tested samples using high resolution mass spectrometry (Exactive-Orbitrap).

Discrimination of Solanaceae taxa and quantification of scopolamine and hyoscyamine by ATR-FTIR spectroscopy.

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

[Simultaneous determination of scopolamine hydrobromide, atropine sulfate, ephedrine hydrochloride and pseudoephedrine hydrochloride in Zhichuanling oral liquid with HPLC].

OBJECTIVE: To establish an HPLC method for determining the contents of scopolamine hydrobromide, atropine sulfate, ephedrine hydrochloride and pseudoephedrine hydrochloride in Zhichuanling oral liquid. METHOD: Agela Durashell RP-C18 (4. 6 mm x250 mm, 5 microm) was adopted, with acetonitrile-sodium phosphate buffer solution (0. 07 mol L-1 sodium phosphate solution with 17.5 mmol L-1 sodium dodecylsulfate adjusted to pH 6.0 with phosphoric acid solution) (30:70) as the mobile phase. The flow rate was 0. 9 mL min -1, the detection wavelength was 207 nm, and the column temperature was 25 degree C. RESULT: Scopolamine hydrobromide, atropine sulfate, ephedrine hlvdrochloride and pseudoephedrine hydrochloride showed good linear relations with peak areas within the concentration range of 0. 021 21-1. 060 5 pg (r =0. 999 3) , 0. 011 14-0. 557 microg (r = 0. 999 6) , 0. 200 56-10. 028 microg (r =0. 999 7) and 0.070 33-3. 516 5 gg (r =0. 999 6), respectively, with the average recoveries of 101.9% , 99. 80%, 100. 3%, 100. 2% (n=6). CONCLUSION: The method was so quick, simple, highly reproducible and specific that it could be used as one of quality control methods of Zhichuanling oral liquid.

Determination of tropane alkaloids atropine and scopolamine by liquid chromatography-mass spectrometry in plant organs of Datura species.

Hyoscyamine (atropine) and scopolamine are the predominant tropane alkaloids in the Datura genus, occurring in all plant organs. The assessment of the alkaloid content of various plant parts is essential from the viewpoint of medical use, but also as a potential risk of toxicity for humans and animals. Therefore, a reliable method for the determination of tropane alkaloid content is of high importance. The present work aimed at the elaboration of a rapid method for determination of the most abundant Datura alkaloids by LC-MS technique using a new generation of core-shell particle packed column. Tropane alkaloid content was investigated in various plant organs of four Datura taxa (D. innoxia, D. metel, D. stramonium, and D. stramonium var. tatula), grown under the same conditions, in two developmental stages. We have developed a rapid LC-MS method for the quantitative determination of atropine and scopolamine, which was successfully applied to quantify the alkaloids in different plant organs (leaves, flowers, stems, seeds) of thorn apples after a simple sample preparation step. Elaboration and validation of the method and analysis of plant extracts were done by UFLC-MS technique, employing an Ascentis Express C18 column. Detection was done in positive ionization mode (ESI+) and the method suitability was evaluated by several validation characteristics. Quantitation limits are 333 and 167 pgmL(-1) for scopolamine and atropine, respectively, and the method shows very good repeatability. The analysis of Datura extracts revealed significant differences depending on the species, the organ and the sampling period. Atropine was found to be dominant over scopolamine in three out of the four taxa investigated. D. innoxia showed the highest concentrations of scopolamine in all organs examined, whereas D. metel accumulated the lowest scopolamine levels. Hyoscyamine, measured as atropine, was the highest in D. stramonium var. tatula, and the lowest in D. innoxia. Samples collected in summer had higher scopolamine levels than autumn samples, concerning both stems and leaves.

A non-aqueous solid phase extraction method for alkaloid enrichment and its application in the determination of hyoscyamine and scopolamine.

A non-aqueous solid phase extraction (SPE) method utilizing silica based strong cation exchange (SCX) was developed and optimized for the enrichment of alkaloids. In this method, silica based SCX SPE columns were used for the elimination of non-alkaloid compounds and the preconcentration of alkaloids from the extracts. Mass spectrometry was employed to analyze the alkaloid-enriched fraction, and results showed that the SPE method developed in this study was effective for the removal of non-alkaloids. Then, this pretreatment method was combined with high performance liquid chromatography for the quantification of scopolamine and hyoscyamine from Scopolia tangutica Maxim. The recoveries of scopolamine and (-)-hyoscyamine were 98.51% and 91.12%, respectively. Relative standard deviation values were 1.4% for scopolamine and 1.6% for (-)-hyoscyamine. The linearity was good in the 0.01-0.8 mg mL(-1) range for hyoscyamine and 0.01-0.4 mg mL(-1) range for scopolamine.

[Scopolamine and hyoscyamine synthesis in hair roots culture of Datura metel].

OBJECTIVE: To establish the hair roots culture system of Datura metel and study the hair roots growth and biosynthesis of scopolamine and hyoscyamine in hair roots culturing system. METHOD: Direct degermed cotyledon of wild D. metel was infected by Agrobacterium tumefaciens strain C58C1 to obtain hair roots. Growth curves and scopolamine and hyoscyamine biosynthesis curves were determined. The scopolamine and hyoscyamine from different hair roots lines were examined by HPLC. RESULT: Hair roots induction rate reached 70%. After 25 days cultured in 1/2 MS liquid nutrient medium, the hair roots weight, content of scopolamine and hyoscyamine reached maximum, tow high efficient accumulation hyoscyamine and scopolamine hair roots lines M1 and M2 were obtained. The medial accumulation coefficient of hyoscyamine and scopolamine were 2.53 times and 5.37 times compared with the leaves of wild D. metel respectively. CONCLUSION: The established hair roots induction and culture system of D. metel provided a foundation for further obtaining scopolamine and hyoscyamine.

Preparative separation of atropine and scopolamine from Daturae metelis Flos using pH-zone-refining counter-current chromatography with counter-rotation and dual-mode elution procedure.

To isolate atropine and scopolamine from Daturae metelis Flos, three different elution modes have been applied in pH-zone-refining counter-current chromatography. These separations were performed with a two-phase solvent system composed of ethyl acetate/n-butanol/water (4:1:5 v/v) with 0.50% triethylamine in the organic phase and 0.15% hydrochloric acid in the aqueous phase. As a result, the best separation was obtained by counter-rotation and dual-mode elution procedure. In this new separation mode, the mobile phase and stationary phase were exchanged when the rotation direction was reversed. The two purified alkaloids (purity over 98% as determined by HPLC) were identified by ESI-MS, (1)H-NMR and (13)C-NMR.