A chromosome-level genome assembly of anesthetic drug-producing Anisodus acutangulus provides insights into its evolution and the biosynthesis of tropane alkaloids.

Tropane alkaloids (TAs), which are anticholinergic agents, are an essential class of natural compounds, and there is a growing demand for TAs with anesthetic, analgesic, and spasmolytic effects. Anisodus acutangulus (Solanaceae) is a TA-producing plant that was used as an anesthetic in ancient China. In this study, we assembled a high-quality, chromosome-scale genome of A. acutangulus with a contig N50 of 7.4 Mb. A recent whole-genome duplication occurred in A. acutangulus after its divergence from other Solanaceae species, which resulted in the duplication of ADC1 and UGT genes involved in TA biosynthesis. The catalytic activities of H6H enzymes were determined for three Solanaceae plants. On the basis of evolution and co-expressed genes, AaWRKY11 was selected for further analyses, which revealed that its encoded transcription factor promotes TA biosynthesis by activating AaH6H1 expression. These findings provide useful insights into genome evolution related to TA biosynthesis and have potential implications for genetic manipulation of TA-producing plants.

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).

Simultaneous quantification of hyoscyamine and scopolamine using HPLC-DAD in four Solanaceae: Hyoscyamus niger, Datura stramonium, Atropa belladonna and Mandragora officinarum.

Alkaloids are a group of secondary metabolites that generate great interest since ancient times. Numerous Solanaceae plants are rich sources of tropane alkaloids as hyoscyamine and scopolamine which are obtained mainly from Hyoscyamus niger, Datura stramonium, Atropa belladonna, Mandragora officinarum. In the present study it was developed an HPLC-DAD using an XBridge Phenyl column for the quantification of scopolamine and hyoscyamine, molecules used in pharmaceutical industry to treat stomach or intestinal disorders. A. belladonna presented hyoscyamine and scopolamine, the first one ranged from 1466 to 5117 mg/Kg DW while the second one ranged from 140 to 1743 mg/Kg DW. In D. stramonium, hyoscyamine was not found while scopolamine ranged from 430 to 8980 mg/Kg DW. On the contrary H. niger and M. officinarum did not contain any trace of these alkaloids. This is the first work in which different parts of four Solanaceae were analysed for their hyoscyamine and scopolamine content.

Metabolic Effects of Elicitors on the Biosynthesis of Tropane Alkaloids in Medicinal Plants.

Tropane alkaloids (TAs) are large secondary metabolite alkaloids that find extensive applications in the synthesis of antidotes, anesthetics, antiemetics, motion sickness drugs, and antispasmodics. The current production method primarily depends on extraction from medicinal plants of the Solanaceae family. Elicitation, as a highly effective biotechnological approach, offers significant advantages in augmenting the synthesis of secondary metabolites. The advantages include its simplicity of operation, low cost, and reduced risk of contamination. This review focuses on the impact of elicitation on the biosynthesis of TAs from three aspects: single-elicitor treatment, multiple-elicitor treatment, and the combination of elicitation strategy with other strategies. Some potential reasons are also proposed. Plant hormones and growth regulators, such as jasmonic acid (JA), salicylic acid (SA), and their derivatives, have been extensively employed in the separate elicitation processes. In recent years, novel elicitors represented by magnetic nanoparticles have emerged as significant factors in the investigation of yield enhancement in TAs. This approach shows promising potential for further development. The current utilization of multi-elicitor treatment is constrained, primarily relying on the combination of only two elicitors for induction. Some of these combinations have been found to exhibit synergistic amplification effects. However, the underlying molecular mechanism responsible for this phenomenon remains largely unknown. The literature concerning the integration of elicitation strategy with other strategies is limited, and several research gaps require further investigation. In conclusion, the impact of various elicitors on the accumulation of TAs is well-documented. However, further research is necessary to effectively implement elicitation strategies in commercial production. This includes the development of stable bioreactors, the elucidation of regulatory mechanisms, and the identification of more potent elicitors.

Hyoscyamine induces developmental toxicity by disrupting metabolism in zebrafish embryo (Danio rerio).

Hyoscyamine is a kind of tropane alkaloids, which exists in several plants of the family Solanaceae. However, the mechanism underlying such hyoscyamine toxic effects during early development remains unclear. In this study, an untargeted metabolomics approach was used to investigate the toxic mechanisms of hyoscyamine in zebrafish embryos. The LC10 and MNLC of hyoscyamine in zebrafish embryos were determined to be 350 and 313 µg/mL, respectively. Moreover, hyoscyamine exposure increased the accumulation of ROS and MDA, and altered the activity of antioxidant enzymes (CAT, SOD, and GSH) in zebrafish embryos. After exposure, the embryos were extracted, derivatized and analyzed by UHPLC-Q-Orbitrap-HRMS for 3551 metabolites to identify 38 significantly changed metabolites based on the VIP, p value, and fold change results. Metabolic pathways associated with those metabolites were identified using MetaboAnalyst 5.0 as follows: pyrimidine metabolism, purine metabolism, histidine metabolism, beta-Alanine metabolism, and glutathione metabolism. These results suggested that hyoscyamine exposure to zebrafish embryos exhibited marked metabolic disturbance. Such significant perturbations of important metabolites within crucial biochemical pathways may have biologically hazardous effects on zebrafish embryos induced by hyoscyamine.

Recycling of hyoscyamine 6ß-hydroxylase for the in vitro production of anisodamine and scopolamine.

The tropane alkaloids hyoscyamine, anisodamine, and scopolamine are extensively used medicines. In particular, scopolamine has the greatest value in the market. Hence, strategies to enhance its production have been explored as an alternative to traditional field-plant cultivation. In this work, we developed biocatalytic strategies for the transformation of hyoscyamine into its products utilizing a recombinant Hyoscyamine 6ß-hydroxylase (H6H) fusion protein to the chitin-binding domain of the chitinase A1 from Bacillus subtilis (ChBD-H6H). Catalysis was carried out in batch, and recycling of H6H constructions was performed via affinity-immobilization, glutaraldehyde crosslinking, and adsorption-desorption of the enzyme to different chitin matrices. ChBD-H6H utilized as free enzyme achieved complete conversion of hyoscyamine in 3- and 22-h bioprocesses. Chitin particles demonstrated to be the most convenient support for ChBD-H6H immobilization and recycling. Affinity-immobilized ChBD-H6H operated in a three-cycle bioprocess (3 h/cycle, 30 °C) yielded in the first and third reaction cycle 49.8% and 22.2% of anisodamine and 0.7% and 0.3% of scopolamine, respectively. However, glutaraldehyde crosslinking decreased enzymatic activity in a broad range of concentrations. Instead, the adsorption-desorption approach equaled the maximal conversion of the free enzyme in the first cycle and retained higher enzymatic activity than the carrier-bound strategy along the consecutive cycles. The adsorption-desorption strategy permitted the reutilization of the enzyme in a simple and economical manner while exploiting the maximal conversion activity displayed by the free enzyme. This approach is valid since other enzymes present in the E. coli lysate do not interfere with the reaction. KEY POINTS: • A biocatalytic system for anisodamine and scopolamine production was developed. • Affinity-immobilized ChBD-H6H in ChP retained catalytic activity. • Enzyme-recycling by adsorption-desorption strategies improves product yields.

Rapid screening of 28 alkaloids in food poisoning samples by liquid chromatography-tandem mass spectrometry / 中国热带医学

@#Abstract: Objective　To investigate a poisoning incident caused by eating eight treasure congee, and establish liquid chromatography (LC)-mass spectrometry (MS)/MS screening method of 28 alkaloids to provide references for disposal of similar poisoning incidents. Methods　LC-MS/MS was used for screening 28 alkaloids in the urine, eight treasure congee and food raw material, and the detected alkaloids were quantified. Samples were extracted with 0.4% formic acid aqueous solution and separated by a Acquity UPLC BEH C18 column (1.7 μm, 100 × 2.1 mm). Acetonitrile-0.2% formic acid aqueous solution was used as the mobile phase and gradient elution was adopted. The ionization mode was electrospray positive ionization mode, and the detection method was multi-reaction monitoring (MRM). Analytes were quantified with the external standard method. Results　In the concentration range of 0-100 ng/mL, the linear correlation coefficient r were greater than 0.999 for 28 alkaloids. The recovery of 28 alkaloids in urine sample ranged from 63.0% to 105.0%, and the relative standard deviations (RSDs) were between 5.8% and 8.6%. The recovery of 28 alkaloids in eight treasure congee sample ranged from 72.0% to 109.0%, and the RSDs were between 6.3% and 9.7%. The recovery of 28 alkaloids in semen sesami nigrum sample ranged from 60.0% to 95.0%, and the RSDs were between 4.8% and 8.2%. Hyoscyamine (2 380.0 ng/mL), scopliamine (3.6 ng/mL) and rac-anisodamine (4.7 ng/mL) were detected in the patient's urine. Hyoscyamine (63.3 μg/g), scopliamine (5.7 μg/g) and rac-anisodamine (2.1 μg/g) were detected in eight treasure congee. Hyoscyamine (901.0 μg/g), scopliamine (80.0 μg/g) and rac-anisodamine (30.1 μg/g) were detected in the seed of Datura stramonium L. The ratio of scopliamine and hyoscyamine in the seed of D. stramonium was 1∶11, which complies with the characteristics of D. stramonium L. In urine sample, the proportion of scopliamine and rac-anisodamine was 0.15% and 0.20%, and hyoscyamine accounted for 99.65%. Conclusion　Seed morphology, the content range and proportion of three alkaloids are all in accord with the characteristics of D. stramonium. Combined with the clinical symptoms of atropine poisoning, it can be deduced that this incident is a family food poisoning caused by accidental consumption of seed of D. stramonium L. The method can provide technical support for the clinical diagnosis and treatment of alkaloid poisoning patients, and also provide a basis for emergency detection and disposal of alkaloid poisoning events.

Effect of the rolB gene on phenotypic development and tropane alkaloids biosynthesis in Atropa belladonna / 药学学报

The rol genes on pRiA4 plasmid of Agrobacterium rhizogenes are potent genes that promote secondary metabolism. Molecular breeding of Atropa belladonna can be conducted by introducing rol genes to increase tropane alkaloids (TAs) content in A. belladonna. In this study, the rolB gene was overexpressed in A. belladonna plants to study the effect of rolB gene on the biosynthesis of TAs. The phenotype, TAs content and expression levels of key enzyme genes in the pathway of TAs biosynthesis of transgenic A. belladonna were analyzed. The results showed that transgenic A. belladonna had developed root system, enlarged leaves, increased leaf fresh weight, deepened leaf color, enlarged flowers, changed flower shape, reduced pistil height and decreased pollen vitality. The content of TAs in the stems of transgenic A. belladonna was significantly higher than that of the control, and the contents of scopolamine, anisodamine, hyoscyamine can reach 2.11-2.91, 1.23-2.37 and 4.88-5.20 times of the control, respectively. Compared with the control group, the expressions of key enzymes putrescine N-methyltransferase (PMT), type III polyketide synthase (PYKS), tropinone reductase I (TRI), aromatic amino acid aminotransferase 4 (ArAT4), UDP-glycosyltransferase 1 (UGT1) and hyoscyamine 6-β-hydroxylase (H6H) in the TAs biosynthesis pathway were up-regulated, and the expression of tropinone reductase II (TRII) as a metabolic shunting gene was down-regulated. The results indicated that rolB gene enhanced TAs synthesis ability in roots and accumulation in stems of A. belladonna by enhancing metabolic flow of TAs synthesis pathway and weakening the metabolic shunt of competing pathway. This study laid a foundation for molecular breeding of A. belladonna with high-yield TAs content using rolB gene.

MIL-53(Fe)-derived ant nest structured porous carbon nanospheres CuFeS2 /C for the determination of atropine enantiomeric impurity L-hyoscyamine.

In this work, an ant nest structured porous carbon nanosphere had been developed for the recognition detection of the atropine (ATP) enantiomers D-hyoscyamine (D-HSM) and L-hyoscyamine (L-HSM). Firstly, Fe-based organic framework was used as the substrate, and Cu ions and sulfur ions were separately introduced to obtain CuFeS2 with ant nest structure by hydrothermal incubation. Then CuFeS2 /C porous nanospheres (PNSs) were obtained by high-temperature calcination. The composite-modified electrode exhibited superior electrochemical performance for L-HSM due to the synergistic effect of CuFeS2 cubic crystals and porous carbon, which has the high specific surface area of the ant nest structure. In addition, the molecularly imprinted polymer (MIP) about L-HSM formed with sulfonated-ß-cyclodextrin (S-ß-CD) and L-arginine (L-Arg) by cyclic voltammetry showed strong chiral recognition of D/L-HSM (ATP). Therefore, a novel electrochemical sensor was constructed based on CuFeS2 /C PNSs and MIP to detect L-HSM by differential pulse voltammetry. Under the optimal conditions, the peak current density of L-HSM showed a good linearity in the concentration range of 0.02-4.6 µM with LOD and LOQ of 0.45 and 1.5 nM, respectively. The oxidation peaks of L-HSM and D-HSM were successfully identified from the racemic ATP, and the oxidation peak potential difference (ΔEp ) between them was 0.138 V. In conclusion, the sensor showed excellent reproducibility, repeatability, and stability and had been applied to the determination of L-HSM in human serum, saliva, and ATP sulfate tablets with satisfactory results.

A validated liquid chromatography-tandem mass spectrometry method for the determination of l-hyoscyamine in human plasma: Application in a clinical study.

Atropine is a racemic mixture of d- and l-hyoscyamine, but only l-hyoscyamine is the effective ingredient. In this study, a new, sensitive, stable, and selective LC/MS assay was developed for the determination of l-hyoscyamine and applied to a clinical study. The parent-product (m/z) transition pair of l-hyoscyamine was 290.1 → 124.1. Chromatographic separations were performed using a chiral MZ column (250 mm × 4.6 mm, 5.0 µm) by a stepwise gradient elution mode with n-hexane, isopropanol, and diethylamine as mobile phases. l-Hyoscyamine in human plasma was extracted by liquid-liquid extraction. This assay displayed a good linearity over a concentration range of 20.0-400 pg/mL for l-hyoscyamine. The accuracy of the validation assay for l-hyoscyamine ranged from -2.7% to 4.5%, and the precision was within 6.3% coefficient of variation. l-Hyoscyamine in human plasma remained stable at different storage conditions. The method has been successfully applied to plasma samples obtained from a safety study in humans.

Improvement on Quality Standard of Belladonnae Herba / 中国实验方剂学杂志

ObjectiveTo improve the current standard of Belladonnae Herba in the 2020 edition of Chinese Pharmacopoeia. MethodTaking hyoscyamine sulfate， atropine sulfate and scopoletin as reference substances， and ethyl acetate-methanol-concentrated ammonia（17∶4∶2）as developing solvent， thin layer chromatography （TLC） was applied in the qualitative identification of Belladonnae Herba. The moisture， total ash and ethanol-soluble extract of Belladonnae Herba were determined based on the general principles in the 2020 edition of Chinese Pharmacopoeia （volume Ⅳ）. The contents of hyoscyamine sulfate and scopolamine hydrobromide were analyzed by high performance liquid chromatography （HPLC） with mobile phase of acetonitrile-54 mmol·L-1 phosphate buffer solution （14∶86）， flow rate of 1.0 mL·min-1 and detection wavelength at 210 nm. ResultThe spots in the TLC were clear with good separation and specificity. Hyoscyamine sulfate and scopolamine hydrobromide showed a good linearity with peak area in the range of 0.024 7-0.789 6 g·L-1 （r=0.999 9） and 0.003 9-0.124 0 g·L-1 （r=0.999 9）， the average recoveries of these two ingredients were 100.29% （RSD 1.6%） and 99.04% （RSD 1.4%）， respectively. The limits for moisture， total ash in Belladonnae Herba should be less than 13.0% and the limit for the ethanol-soluble extract should be more than 10.0%. Due to the low content and wide variation of scopolamine hydrobromide， the content of hyoscyamine sulfate should not be less than 0.098%. ConclusionThe established method is simple， specific and reproducible， which can be used to improve the quality control standard of Belladonnae Herba.

Effect of the rolC gene on biosynthesis of tropane alkaloids in Atropa belladonna / 药学学报

Hair roots induced by Agrobacterium rhizogene produce higher levels of secondary metabolites than non-induced plants, and the enhanced metabolic capacity is driven by the rol gene. We hypothesized that rol genes can be utilized to improve the biosynthesis of tropane alkaloids (TAs) in Atropa belladonna. In this study, the rolC gene from Agrobacterium rhizogene pRiA4 plasmid, driven by a CaMV35S promoter, was overexpressed in A. belladonna. The phenotypes, TAs content and transcriptional expression of key genes in TAs biosynthesis were analyzed in transgenic A. belladonna plants. Results show that transgenic A. belladonna exhibited a well-developed root system, male sterility, higher stamen column length than pistil, early flowering, internode shortening, smaller but more flowers, increased axillary buds and lateral buds, decreased apical dominance, and long and narrow leaves as compared to wild-type plants. Transgenic A. belladonna produced more TAs than wild-type plants, with the content of hyoscyamine, anisodamine and scopolamine reaching 2.58, 3.59 and 15.77-fold that of the control group, respectively. The gene expression of putrescine N-methyltransferase (PMT), tropinone reductase I (TRⅠ) and hyoscyamine 6-β-hydroxylase (H6H), key enzymes in TAs biosynthesis, were up-regulated compared with the control group. The above results indicate that the rolC gene enhances TAs biosynthesis in A. belladonna by up-regulating the expression of key enzymes in the TAs biosynthesis pathway, laying a foundation for genetic manipulation of A. belladonna to increase TAs content by increasing rolC gene expression.

Pharmacokinetics and Effects on Saliva Flow of Sublingual and Oral Atropine in Clozapine-Treated and Healthy Adults: An Interventional Cross-Over Study.

Background: Sublingual atropine is an effective treatment of clozapine-induced hypersalivation. This study aims to investigate the pharmacokinetics of atropine after sublingual and oral administration and study the dose effect of atropine on saliva secretion. Methods: An interventional cross-over clinical trial where participants received 0.6 mg and 1.2 mg atropine sulfate sublingual solution and 0.6 mg oral tablet. Atropine plasma concentration was measured over 9 hours with validated LC-MS/MS method. Atropine effects on saliva secretion rate, visual acuity and accommodation, and vital signs were assessed. Results: Four clozapine-treated and three healthy participants were enrolled in the study. The area under the atropine plasma concentration-time curve (AUC0-∞) was highest after the 1.2 mg sublingual solution administration in comparison with 0.6 mg tablet or sublingual solution (8.58±1.66 µg.L-1.h vs. 4.65±1.29 vs. 2.98±0.73 µg.L-1.h, respectively). The Cmax for the 0.6 mg and 1.2 mg sublingual solutions was 1.11±0.99 and 1.76±0.62 µg.L-1, and tmax was 2.18±0.59 and 1.9±0.71 h, respectively. In comparison with the 0.6 mg sublingual solution dose, the saliva secretion reduction was larger after the oral tablet administration (-40% (-59, -22%) vs. -69% (-80, -57)) and largest after the 1.2 mg sublingual solution administration (-79% (-93,-64)). Conclusion: Both the sublingual and oral atropine are effective in reducing the saliva secretion however at a lower plasma concentration after sublingual administration, with a dose-dependent effect. Both have significantly reduced the blood pressure and pulse rate over 3 hours without significant changes in vision. No major safety concerns were reported.

Structural insights on the interaction potential of natural leads against major protein targets of SARS-CoV-2: Molecular modelling, docking and dynamic simulation studies.

Though significant efforts are in progress for developing drugs and vaccines against COVID-19, limited therapeutic agents are available currently. Thus, it is essential to undertake COVID-19 research and to identify therapeutic interventions in which computational modeling and virtual screening of lead molecules provide significant insights. The present study aimed to predict the interaction potential of natural lead molecules against prospective protein targets of SARS-CoV-2 by molecular modeling, docking, and dynamic simulation. Based on the literature survey and database search, fourteen molecular targets were selected and the three targets which lack the native structures were computationally modeled. The drug-likeliness and pharmacokinetic features of ninety-two natural molecules were predicted. Four lead molecules with ideal drug-likeliness and pharmacokinetic properties were selected and docked against fourteen targets, and their binding energies were compared with the binding energy of the interaction between Chloroquine and Hydroxychloroquine to their usual targets. The stabilities of selected docked complexes were confirmed by MD simulation and energy calculations. Four natural molecules demonstrated profound binding to most of the prioritized targets, especially, Hyoscyamine and Tamaridone to spike glycoprotein and Rotiorinol-C and Scutifoliamide-A to replicase polyprotein-1ab main protease of SARS-CoV-2 showed better binding energy, conformational and dynamic stabilities compared to the binding energy of Chloroquine and its usual target glutathione-S-transferase. The aforementioned lead molecules can be used to develop novel therapeutic agents towards the protein targets of SARS-CoV-2, and the study provides significant insight for structure-based drug development against COVID-19.

Development of Atropa belladonna L. Plants with High-Yield Hyoscyamine and without Its Derivatives Using the CRISPR/Cas9 System.

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

Engineered Microbes for Producing Anticholinergics.

The tropane alkaloids (TAs) hyoscyamine and scopolamine function as acetylcholine receptor antagonists and are used clinically as parasympatholytics to treat neuromuscular disorders in humans. While TAs are synthesized in a small subset of plant families, these specialized metabolites are only accumulated in limited quantities in plant organs. The complex chemical structures of these compounds make their industrial production by chemical synthesis very challenging, Therefore, the supply of these TAs still relies on intensive farming of Duboisia shrubs in tropical countries. Many adverse factors such as climate fluctuations and pandemics can thus influence annual world production. Based on the landmark microbial production of the antimalarial semi-synthetic artemisinin, the Smolke group recently developed a yeast cell factory capable of de novo synthesizing hyoscyamine and scopolamine, thus paving the way for an alternative production of these compounds.

Improved tropane alkaloid production and changes in gene expression in hairy root cultures of two Hyoscyamus species elicited by silicon dioxide nanoparticles.

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.

Molecular cloning and functional analysis of hyoscyamine 6ß-hydroxylase (H6H) in the poisonous and medicinal plant Datura innoxia mill.

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