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).

Multiple independent losses of the biosynthetic pathway for two tropane alkaloids in the Solanaceae family.

Hyoscyamine and scopolamine (HS), two valuable tropane alkaloids of significant medicinal importance, are found in multiple distantly related lineages within the Solanaceae family. Here we sequence the genomes of three representative species that produce HS from these lineages, and one species that does not produce HS. Our analysis reveals a shared biosynthetic pathway responsible for HS production in the three HS-producing species. We observe a high level of gene collinearity related to HS synthesis across the family in both types of species. By introducing gain-of-function and loss-of-function mutations at key sites, we confirm the reduced/lost or re-activated functions of critical genes involved in HS synthesis in both types of species, respectively. These findings indicate independent and repeated losses of the HS biosynthesis pathway since its origin in the ancestral lineage. Our results hold promise for potential future applications in the artificial engineering of HS biosynthesis in Solanaceae crops.

Viral inhibitory potential of hyoscyamine in Japanese encephalitis virus-infected embryonated chicken eggs involving multiple signaling pathways.

Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis worldwide. The emergence of new genotypes of the virus and a high rate of mutation make it necessary to develop alternative treatment strategies against this deadly pathogen. Although the antiviral properties of Atropa belladonna and some of its active components, such as atropine and scopolamine, have been studied, the effect of another important component, hyoscyamine, against JEV infection has not yet been investigated. In this study, we investigated the antiviral effect of hyoscyamine against JEV and its immunomodulatory activity in embryonated chicken eggs. Pretreatment with hyoscyamine sulphate resulted in a significant decrease in the viral load in both chorioallantoic membrane (CAM) and brain tissues at 48 and 96 hours postinfection. In silico studies showed stable binding and interaction between hyoscyamine and non-structural protein 5 (NS5), suggesting that this could be the basis of its antiviral effect. Embryonated eggs pretreated with hyoscyamine sulphate showed upregulation of Toll-like receptor 3 (TLR3), TLR7, TLR8, interleukin 4 (IL-4), and IL-10 as well as interferons and regulatory factors. Hyoscyamine sulphate was also found to cause significant downregulation of TLR4. The potential use of hyoscyamine for controlling JEV replication and its dissemination to the brain suggest that it may be a promising therapy option against JEV in the future.

Tropinone reductase: A comprehensive review on its role as the key enzyme in tropane alkaloids biosynthesis.

TAs, including hyoscyamine and scopolamine, were used to treat neuromuscular disorders ranging from nerve agent poisoning to Parkinson's disease. Tropinone reductase I (TR-I; EC 1.1.1.206) catalyzed the conversion of tropinone into tropine in the biosynthesis of TAs, directing the metabolic flow towards hyoscyamine and scopolamine. Tropinone reductase II (TR-II; EC 1.1.1.236) was responsible for the conversion of tropinone into pseudotropine, diverting the metabolic flux towards calystegine A3. The regulation of metabolite flow through both branches of the TAs pathway seemed to be influenced by the enzymatic activity of both enzymes and their accessibility to the precursor tropinone. The significant interest in the utilization of metabolic engineering for the efficient production of TAs has highlighted the importance of TRs as crucial enzymes that govern both the direction of metabolic flow and the yield of products. This review discussed recent advances for the TRs sources, properties, protein structure and biocatalytic mechanisms, and a detailed overview of its crucial role in the metabolism and synthesis of TAs was summarized. Furthermore, we conducted a detailed investigation into the evolutionary origins of these two TRs. A prospective analysis of potential challenges and applications of TRs was presented.

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.

The Evolutionary Pattern of Cocaine and Hyoscyamine Biosynthesis Provides Strategies To Produce Tropane Alkaloids.

Cocaine and hyoscyamine are two tropane alkaloids (TA) from Erythroxylaceae and Solanaceae, respectively. These famous compounds possess anticholinergic properties that can be used to treat neuromuscular disorders. While the hyoscyamine biosynthetic pathway has been fully elucidated allowing its de novo synthesis in yeast, the cocaine pathway remained only partially elucidated. Recently, the Huang research group has completed the cocaine biosynthetic route by characterizing its two missing enzymes. This allowed the whole pathway to be transferring into Nicotiana benthamiana to achieve cocaine production. Here, besides highlighting the impact of this discovery, we discuss how TA biosynthesis evolved via the recruitment of two distinct and convergent pathways in Erythroxylaceae and Solanaceae. Finally, while enriching our knowledge on TA biosynthesis, this diversification of the molecular actors involved in cocaine and hyoscyamine biosynthesis opens perspectives in metabolic engineering by exploring enzyme biochemical plasticity that can ease and shorten TA pathway reconstitution in heterologous organisms.

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.

Genomic and structural basis for evolution of tropane alkaloid biosynthesis.

The tropane alkaloids (TAs) cocaine and hyoscyamine have been used medicinally for thousands of years. To understand the evolutionary origins and trajectories of serial biosynthetic enzymes of TAs and especially the characteristic tropane skeletons, we generated the chromosome-level genome assemblies of cocaine-producing Erythroxylum novogranatense (Erythroxylaceae, rosids clade) and hyoscyamine-producing Anisodus acutangulus (Solanaceae, asterids clade). Comparative genomic and phylogenetic analysis suggested that the lack of spermidine synthase/N-methyltransferase (EnSPMT1) in ancestral asterids species contributed to the divergence of polyamine (spermidine or putrescine) methylation in cocaine and hyoscyamine biosynthesis. Molecular docking analysis and key site mutation experiments suggested that ecgonone synthases CYP81AN15 and CYP82M3 adopt different active-site architectures to biosynthesize the same product ecgonone from the same substrate in Erythroxylaceae and Solanaceae. Further synteny analysis showed different evolutionary origins and trajectories of CYP81AN15 and CYP82M3, particularly the emergence of CYP81AN15 through the neofunctionalization of ancient tandem duplication genes. The combination of structural biology and comparative genomic analysis revealed that ecgonone methyltransferase, which is responsible for the biosynthesis of characteristic 2-substituted carboxymethyl group in cocaine, evolved from the tandem copies of salicylic acid methyltransferase by the mutations of critical E216 and S153 residues. Overall, we provided strong evidence for the independent origins of serial TA biosynthetic enzymes on the genomic and structural level, underlying the chemotypic convergence of TAs in phylogenetically distant species.

Revealing evolution of tropane alkaloid biosynthesis by analyzing two genomes in the Solanaceae family.

Tropane alkaloids (TAs) are widely distributed in the Solanaceae, while some important medicinal tropane alkaloids (mTAs), such as hyoscyamine and scopolamine, are restricted to certain species/tribes in this family. Little is known about the genomic basis and evolution of TAs biosynthesis and specialization in the Solanaceae. Here, we present chromosome-level genomes of two representative mTAs-producing species: Atropa belladonna and Datura stramonium. Our results reveal that the two species employ a conserved biosynthetic pathway to produce mTAs despite being distantly related within the nightshade family. A conserved gene cluster combined with gene duplication underlies the wide distribution of TAs in this family. We also provide evidence that branching genes leading to mTAs likely have evolved in early ancestral Solanaceae species but have been lost in most of the lineages, with A. belladonna and D. stramonium being exceptions. Furthermore, we identify a cytochrome P450 that modifies hyoscyamine into norhyoscyamine. Our results provide a genomic basis for evolutionary insights into the biosynthesis of TAs in the Solanaceae and will be useful for biotechnological production of mTAs via synthetic biology approaches.

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.

Catalytic innovation underlies independent recruitment of polyketide synthases in cocaine and hyoscyamine biosynthesis.

Tropane alkaloids such as hyoscyamine and cocaine are of importance in medicinal uses. Only recently has the hyoscyamine biosynthetic machinery become complete. However, the cocaine biosynthesis pathway remains only partially elucidated. Here we characterize polyketide synthases required for generating 3-oxo-glutaric acid from malonyl-CoA in cocaine biosynthetic route. Structural analysis shows that these two polyketide synthases adopt distinctly different active site architecture to catalyze the same reaction as pyrrolidine ketide synthase in hyoscyamine biosynthesis, revealing an unusual parallel/convergent evolution of biochemical function in homologous enzymes. Further phylogenetic analysis suggests lineage-specific acquisition of polyketide synthases required for tropane alkaloid biosynthesis in Erythroxylaceae and Solanaceae species, respectively. Overall, our work elucidates not only a key unknown step in cocaine biosynthesis pathway but also, more importantly, structural and biochemical basis for independent recruitment of polyketide synthases in tropane alkaloid biosynthesis, thus broadening the understanding of conservation and innovation of biosynthetic catalysts.

An organic transistor for the selective detection of tropane alkaloids utilizing a molecularly imprinted polymer.

This study proposes a chemical sensing approach for the selective detection of tropane alkaloid drugs based on an extended-gate-type organic field-effect transistor (OFET) functionalized with a molecularly imprinted polymer (MIP). From the viewpoint of pharmaceutical chemistry, the development of versatile chemical sensors to determine the enantiomeric purity of over-the-counter (OTC) tropane drugs is important because of their side effects and different pharmacological activities depending on their chirality. To this end, we newly designed an OFET sensor with an MIP (MIP-OFET) as the recognition element for tropane drugs based on a high complementarity among a template (i.e., (S)-hyoscyamine) and functional monomers such as N-isopropylacrylamide and 2,2-dimethyl-4-pentenoic acid. Indeed, the MIP optimized by density functional theory (DFT) has succeeded in the sensitive and selective detection of (S)-hyoscyamine (as low as 1 µM) by the combination of the OFET with highly selective recognition sites in the MIP. The MIP-OFET was further applied to determine the enantiomeric excess (ee) of commercially available (S)-hyoscyamine, and the linearity changes in the threshold voltages of the OFET corresponded to the % ee values of (S)-hyoscyamine. Overall, the validation with tropane alkaloids revealed the potential of the MIP combined with OFET as a chemical sensor chip for OTC drugs in real-world scenarios.

Engineering tropane alkaloid production and glyphosate resistance by overexpressing AbCaM1 and G2-EPSPS in Atropa belladonna.

Atropa belladonna is an important industrial crop for producing anticholinergic tropane alkaloids (TAs). Using glyphosate as selection pressure, transgenic homozygous plants of A. belladonna are generated, in which a novel calmodulin gene (AbCaM1) and a reported EPSPS gene (G2-EPSPS) are co-overexpressed. AbCaM1 is highly expressed in secondary roots of A. belladonna and has calcium-binding activity. Three transgenic homozygous lines were generated and their glyphosate tolerance and TAs' production were evaluated in the field. Transgenic homozygous lines produced TAs at much higher levels than wild-type plants. In the leaves of T2GC02, T2GC05, and T2GC06, the hyoscyamine content was 8.95-, 10.61-, and 9.96 mg/g DW, the scopolamine content was 1.34-, 1.50- and 0.86 mg/g DW, respectively. Wild-type plants of A. belladonna produced hyoscyamine and scopolamine respectively at the levels of 2.45 mg/g DW and 0.30 mg/g DW in leaves. Gene expression analysis indicated that AbCaM1 significantly up-regulated seven key TA biosynthesis genes. Transgenic homozygous lines could tolerate a commercial recommended dose of glyphosate in the field. In summary, new varieties of A. belladonna not only produce pharmaceutical TAs at high levels but tolerate glyphosate, facilitating industrial production of TAs and weed management at a much lower cost.

Fluorescence-based immunochromatographic test strip for the detection of hyoscyamine.

Hyoscyamine (HSM), which acts as an antagonist of the acetylcholine muscarinic receptor and can induce a variety of distinct toxic syndromes in mammals (anti-cholinergic poisoning), is hazardous to human health. Therefore, it is urgent to develop a rapid, sensitive, and cost-effective method to determine HSM. A fluorescent microsphere based immunochromatographic assay was developed for this analyte and gold nanoparticles (AuNPs) were used as a comparison. A monoclonal antibody against HSM was prepared with a 50% inhibition concentration (IC50) of 1.17 ng mL-1, with no cross-reactivity with five drugs. Under optimized conditions, the cut off limits using the fluorescence-labeled monoclonal antibody strips were 10 ng mL-1 in 0.01 M PBS and 20 ng mL-1 in pork, pig urine, and honey samples, and the assay could be completed within 10 min. In comparison with a AuNP immunochromatographic assay, the developed method offered a higher coupling rate and lower amounts of antibodies. This approach could be used for simple, sensitive and rapid screening, and is suitable for on-site screening applications.

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.

Tropane alkaloid contamination of agricultural commodities and food products in relation to consumer health: Learnings from the 2019 Uganda food aid outbreak.

Tropane alkaloids (TAs) are secondary plant metabolites derived mainly from Solanaceae plant families, with the most virulent invasive species being Datura stramonium. Datura stramonium commonly grows in cereal fields and produce TAs (e.g., hyoscyamine and scopolamine) which may accidentally contaminate cereals (and cereal-based foods) at occasionally high levels. Dietary exposure to TAs can be toxic and depending on the dose ingested can cause outcomes ranging from anticholinergic effects to acute poisoning and death. In 2019, 315 adults became ill and another five adults died in Uganda following consumption of a "Super Cereal" (a fortified blended food) that was later confirmed to be contaminated by TAs-a scenario which provoked this holistic review on TAs in foodstuffs. Thus, this article provides information on the history, development, occurrences, exposures, and human legislative and health benchmarks for TAs. It describes control strategies for reducing TA contamination of agricultural commodities and resultant health implications following consumption of TA contaminated foodstuffs. Adequate application of food safety control measures (including maximum limits) and good practices, from the start of cereal cultivation through to the final stages of manufacturing of food products can aid in the reduction of seeing toxic plants including D. stramonium in cereal fields.

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.

Biosynthesis of medicinal tropane alkaloids in yeast.

Tropane alkaloids from nightshade plants are neurotransmitter inhibitors that are used for treating neuromuscular disorders and are classified as essential medicines by the World Health Organization1,2. Challenges in global supplies have resulted in frequent shortages of these drugs3,4. Further vulnerabilities in supply chains have been revealed by events such as the Australian wildfires5 and the COVID-19 pandemic6. Rapidly deployable production strategies that are robust to environmental and socioeconomic upheaval7,8 are needed. Here we engineered baker's yeast to produce the medicinal alkaloids hyoscyamine and scopolamine, starting from simple sugars and amino acids. We combined functional genomics to identify a missing pathway enzyme, protein engineering to enable the functional expression of an acyltransferase via trafficking to the vacuole, heterologous transporters to facilitate intracellular routing, and strain optimization to improve titres. Our integrated system positions more than twenty proteins adapted from yeast, bacteria, plants and animals across six sub-cellular locations to recapitulate the spatial organization of tropane alkaloid biosynthesis in plants. Microbial biosynthesis platforms can facilitate the discovery of tropane alkaloid derivatives as new therapeutic agents for neurological disease and, once scaled, enable robust and agile supply of these essential medicines.

Promoting the accumulation of scopolamine and hyoscyamine in Hyoscyamus niger L. through EMS based mutagenesis.

The overexploitation of medicinal plants is depleting gene pool at an alarming rate. In this scenario inducing the genetic variability through targeted mutations could be beneficial in generating varieties with increased content of active compounds. The present study aimed to develop a reproducible protocol for in vitro multiplication and mutagenesis of Hyoscyamus niger targeting putrescine N-methyltransferase (PMT) and 6ß-hydroxy hyoscyamine (H6H) genes of alkaloid biosynthetic pathway. In vitro raised callus were treated with different concentrations (0.01% - 0.1%) of Ethyl Methane Sulfonate (EMS). Emerging multiple shoots and roots were obtained on the MS media supplemented with cytokinins and auxins. Significant effects on morphological characteristics were observed following exposure to different concentrations of EMS. EMS at a concentration of 0.03% was seen to be effective in enhancing the average shoot and root number from 14.5±0.30 to 22.2 ±0.77 and 7.2±0.12 to 8.8±0.72, respectively. The lethal dose (LD50) dose was calculated at 0.08% EMS. The results depicted that EMS has an intense effect on PMT and H6H gene expression and metabolite accumulation. The transcripts of PMT and H6H were significantly upregulated at 0.03-0.05% EMS compared to control. EMS treated explants showed increased accumulation of scopolamine (0.639 µg/g) and hyoscyamine (0.0344µg/g) compared to untreated.