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The use of herbal medicines is expanding rapidly, and reputable pharmaceutical companies are increasing the proportion of effective herbal compounds in the production of drugs used to treat conditions such as cancer and neurological disorders. The commercial-scale production of these natural metabolites by plants remains limited, necessitating the application of modern technologies to enhance their yield. In the present study, the effects of various concentrations of silicon dioxide nanoparticles (SiO₂ NPs) as an elicitor (0, 100, and 200 mg/L) and different precursors (no precursor, phenylalanine, and putrescine at 3 mM) on the growth and phytochemical traits of Hyoscyamus reticulatus hairy roots were investigated. Hairy roots gradually appeared from the wound sites two weeks after inoculation. The highest fresh weight (FW) (3.57 g) was observed in the putrescine precursor treatment, while the maximum dry weight (DW) (0.73 g) was recorded in the control group. The highest total phenolic content (TPC) (7.79 mg GA/g FW) was observed with the putrescine precursor, while the highest total flavonoid content (TFC). (1.79 mg QUE/g FW) was obtained with the phenylalanine precursor, both in the absence of the elicitor. HPLC analysis revealed that the highest levels of hyoscyamine (165.4 µg/g DW) and scopolamine (91.3 µg/g DW) were detected in hairy roots treated with 200 mg/L SiO₂ NPs combined with putrescine precursor. These findings suggest that the combined use of elicitors and precursor feeding in transgenic hairy root cultures of H. reticulatus can significantly enhance the production of tropane alkaloids, such as hyoscyamine and scopolamine.

Hyoscyamus reticulatus L. is a rich source of tropane alkaloids (TA) which is widely used in medicine. In this study, hairy roots were obtained from two-week cotyledon explants of H. reticulatus L. by using the A7 strain of Agrobacterium rhizogenes . The effects of different concentrations of chitosan nanoparticles (CNP) (0, 10, 30, 50 and 100 mg L − 1 ) as a signaling molecule with two exposure times (24 and 48 h) on the growth rate, antioxidant enzymes activity, phenol content, TA contents and hyoscyamine-6-beta-hydroxylase ( h6h ) gene expression levels were investigated. After extraction of TA from hairy roots, High-performance liquid chromatography (HPLC) was used to measure the amount of hyoscyamine and scopolamine. The results showed that the highest amount of hyoscyamine (1300.3 µg g − 1 FW) and scopolamine (918.06 µg g − 1 FW) accumulated in hairy roots treated with 100 mg L − 1 CNP at 48 h and 10 mg L − 1 CNP at 24 h, respectively. In the present study, the effect of CNP on the expression of the h6h gene was also investigated using semi-quantitative RT-PCR. The highest expression of this gene was observed at a concentration of 10 mg L − 1 in 24 h and the lowest was observed in the control sample. Based on these results, it is concluded that CNP can be used as an effective stimulant for the production of plant secondary metabolites, including TA, probably due to the stimulation of gene expression effective in the production of TA.

Chemical synthesis issue of tropane alkaloids and their lowest production rate along with their extraction costs has made use of new methods necessary to produce valuable pharmaceutical compounds. hairy roots induction end elicitation with biotic or abiotic elicitors such as nanoparticles can be used as a source for high production of worth metabolites compared to intact plants. Hyoscyamus reticulatus L. is a rich source of hyoscyamine and scopolamine, which are widely used in pharmaceuticals. Since the in this research, the effect of different concentrations (0, 25, 50, 75 and 100 mg/l) of zinc Nano oxide (ZnO NPs) as a stimulant on the growth rate, antioxidant enzyme activity and antioxidant activity, total phenol content (TPC), total flavonoid content (TFC) and tropane alkaloids in the H. reticulatus hairy roots was investigated in a bubble column bioreactor. The highest and the lowest amount of fresh weight (FW) (153.4 and 79.82 g) were recorded in 50 mg/l during 48 h exposure time and 100 mg/l during 72 h respectively. Also, ANOVA results showed that the interaction effect of concentration and exposure time was significant on guaiacol peroxidase (GPX) enzyme activity, TPC and TFC, antioxidant activity and alkaloid content ( p  < 0.01). HPLC analysis revealed that the highest amount of hyoscyamine and scopolamine (400 and 275.1 mg/kg of DW) were observed at ZnO NPs (75 mg/l) during 72 h respectively. According to the results, it can be said that the use of nano zinc oxide as a stimulant is an effective method for increasing tropane alkaloids in a bubble column bioreactor.

Background The production of tropane alkaloids such as atropine and scopolamine by chemical methods is costly. This study aimed to find a more efficient method for producing tropane alkaloids by investigating different LED light qualities and glycine treatment using leaf callus cultures of Hyoscyamus niger L. in vitro. Results In this study, after producing healthy plantlets, callus induction was done in the B5 medium with 2–4-D ( 2 , 4 -Dichlorophenoxyacetic acid) at 2 mg L −1 with BA (6-benzyl adenine) at a concentration of 1 mg L −1 . The use of LED light and glycine treatments started from the callus stage, in such a way that the callus produced was placed in culture media containing different concentrations of glycine (0, 2, 4 and 8 mg L −1 ). They were transferred to light boxes, and light treatments were applied at 4 levels (darkness (D), white LED light (WLED), blue LED light (BLED), and red LED light (RLED)). The results demonstrate that, glycine had a significant impact on the accumulation of total carbohydrates and total amino acids, quercetin, total phenols and total flavonoids in H. niger L. callus under LED light qualities. The highest atropine was obtained under Blue LED (BLED) conditions with the glycine at mg L −1 , while the highest scopolamine was observed under BLED conditions with glycine at 4 mg L −1 . Also, the highest gallic acid levels (4.18 mg g −1 ) were observed in callus grown under BLED and RED LED (RLED) light conditions with glycine at 8 mg L −1 . Conclusion In the present study, we have developed an efficient protocol for the production of tropane alkaloids by investigating different LED light colors and glycine treatments exploiting in vitro callus cultures of H.niger . These findings could have potential applications in the pharmaceutical and food industries for efficiently producing high-valued alkaloids from the plants.

Key Message: Tetraploidy improves overexpression of h6h and scopolamine production of H. muticus, while in H. senecionis, pmt overexpression and elicitation can be used as effective methods for increasing tropane alkaloids. Abstract: The effects of metabolic engineering in a polyploid context were studied by overexpression of h6h in the tetraploid hairy root cultures of H. muticus. Flow cytometry analysis indicated genetic stability in the majority of the clones, while only a few clones showed genetic instability. Among all the diploid and tetraploid clones, the highest level of h6h transgene expression and scopolamine accumulation was interestingly observed in the tetraploid clones of H. muticus. Therefore, metabolic engineering of the tropane biosynthetic pathway in polyploids is suggested as a potential system for increasing the production of tropane alkaloids. Transgenic hairy root cultures of Hyoscyamus senecionis were also established. While overexpression of pmt in H. senecionis was correlated with a sharp increase in hyoscyamine production, the h6h-overexpressing clones were not able to accumulate higher levels of scopolamine than the leaves of intact plants. Applying methyl jasmonate was followed by a sharp increase in the expression of pmt and a drop in the expression of tropinone reductase II (trII) which consequently resulted in the higher biosynthesis of hyoscyamine and total alkaloids in H. senecionis.

Throughout molecular evolution, organisms create assorted chemicals in response to varying ecological niches. Catalytic landscapes underlie metabolic evolution, wherein mutational steps alter the biosynthetic properties of enzymes. Here we report the first systematic quantitative characterization of the catalytic landscape underlying the evolution of sesquiterpene chemical diversity. On the basis of our previous discovery of a set of nine naturally occurring amino acid substitutions that functionally interconverted orthologous sesquiterpene synthases from Nicotiana tabacum and Hyoscyamus muticus , we created a library of all possible residue combinations (2 9 = 512) in the N. tabacum enzyme. The product spectra of 418 active enzymes revealed a rugged landscape where several minimal combinations of the nine mutations encode convergent solutions to the interconversions of parental activities. Quantitative comparisons indicated context dependence for mutational effects—epistasis—in product specificity and promiscuity. These results provide a measure of the mutational accessibility of phenotypic variability in a diverging lineage of terpene synthases.

This study aimed to investigate the effects of clinorotation induced by 2-D clinostat on the growth, tropane alkaloid production, gene expression, antioxidant capacity, and cellular defense responses in the callus tissue of Hyoscyamus niger. Callus induction was conducted by putting hypocotyl explants in the MS culture medium supplemented with 1 mgL−1 2,4-D and 1 mgL−1 BAP growth regulators. The sub-cultured calli were placed on a clinostat for 0, 3, 7, and 10 days (2.24 × 10–5 g on the edge of the callus ring). Clinorotation significantly increased callus fresh weight, dry weight, protein, carbohydrate, and proline contents compared to the control, and their maximum contents were obtained after 7 and 10 days. H2O2 level enhanced under clinorotation with a 76.3% rise after 10 days compared to control and positively affected the atropine (77.1%) and scopolamine (69.2%) productions. Hyoscyamine 6-beta hydroxylase and putrescine N-methyltransferase gene expression involved in the tropane alkaloid biosynthesis were upregulated markedly with 14.2 and 17.1-folds increase after 10 days of clinorotation, respectively. The expressions of jasmonic acid, mitogen-activated protein kinase, and ethylene-responsive element-binding transcription factor were upregulated, and the activity of peroxidase and catalase showed a 72.7 and 80% rise after 10 days. These findings suggest that microgravity can enhance callogenesis by stimulating the ROS level, which can impact the antioxidant enzymes, tropane alkaloid formation, and gene expression.

The medicinal plant Hyoscyamus reticulatus L. is a rich source of hyoscyamine and scopolamine, the tropane alkaloids. The use of hairy root cultures has focused significant attention on production of important metabolites such as stable tropane alkaloid production. Elicitation is an effective approach to induce secondary metabolite biosynthetic pathways. Hairy roots were derived from cotyledon explants inoculated with Agrobacterium rhizogenes and elicited by iron oxide nanoparticles (FeNPs) at different concentrations (0, 450, 900, 1800, and 3600 mg L−1) for different exposure times (24, 48, and 72 h). The highest hairy root fresh and dry weights were found in the medium supplemented with 900 mg L−1 FeNPs. Antioxidant enzyme activity was significantly increased in induced hairy roots compared to non-transgenic roots. The highest hyoscyamine and scopolamine production (about fivefold increase over the control) was achieved with 900 and 450 mg L−1 FeNPs at 24 and 48 h of exposure time, respectively. This is the first report of the effect of FeNP elicitor on hairy root cultures of a medicinal plant. We suggest that FeNPs could be an effective elicitor in hairy root cultures in order to increase tropane alkaloid production.

This study aimed to investigate the chemical composition and antidiabetic properties of cultivated Hyoscyamus albus L. The ethanol extract was analyzed using LC-MS/MS, and 18 distinct phenolic compounds were identified. Among these, p-coumaric acid (6656.8 ± 3.4 µg/g), gallic acid (6516 ± 1.7 µg/g), luteolin (6251.9 ± 1.3 µg/g), apigenin (6209.9 ± 1.1 µg/g), and rutin (5213.9 ± 1.3 µg/g) were identified as the most abundant polyphenolic molecules. In the in vitro antidiabetic experiment, the ability of the plant extract to inhibit α-glucosidase and α-amylase activities was examined. The results indicated that the extract from H. albus L. exhibited a higher inhibitory effect on α-amylase compared to α-glucosidase, with an IC50 of 146.63 ± 1.1 µg/mL and 270.43 ± 1.1 µg/mL, respectively. Docking simulations revealed that luteolin, fisetin, and rutin exhibited the most promising inhibitory activity against both enzymes, as indicated by their high contrasting inhibition scores. To further investigate the in vivo antidiabetic effects of H. albus L., an experiment was conducted using STZ-induced diabetic mice. The results demonstrated that the plant extract effectively reduced the levels of cholesterol and triglycerides. These findings suggest that H. albus L. may have therapeutic potential for managing hyperlipidemia, a common complication associated with diabetes. This highlights its potential as a natural remedy for diabetes and related conditions.

The tribe Hyoscyameae (Solanaceae) is restricted to Eurasia and includes the genera Archihyoscyamus, Anisodus, Atropa, Atropanthe, Hyoscyamus, Physochlaina, Przewalskia and Scopolia. Even though the monophyly of Hyoscyameae is strongly supported, the relationships of the taxa within the tribe remain unclear. Chloroplast markers have been widely used to elucidate plant relationships at low taxonomic levels. Identification of variable chloroplast intergenic regions has been developed based on comparative genomics of chloroplast genomes, but these regions have a narrow phylogenetic utility. In this study, we present the chloroplast genome sequence of Hyoscyamus niger and make comparisons to other solanaceous plastid genomes in terms of gene order, gene and intron content, editing sites, origins of replication, repeats, and hypothetical open reading frames. We developed and sequenced three variable plastid markers from eight species to elucidate relationships within the tribe Hyoscyameae. The presence of a horizontally transferred intron in the mitochondrial cox1 gene of some species of the tribe is considered here a likely synapomorphy uniting five genera of the Hyoscyameae. Alternatively, the cox1 intron could be a homoplasious character acquired twice within the tribe. A homoplasious inversion in the intergenic plastid spacer trnC-psbM was recognized as a source of bias and removed from the data set used in the phylogenetic analyses. Almost 12 kb of plastid sequence data were not sufficient to completely resolve relationships among genera of Hyoscyameae but some clades were identified. Two alternative hypotheses of the evolution of the genera within the tribe are proposed.