- Biotic elicitation for scopolamine production by hairy root cultures of Datura metel. [Journal Article]
- MBMol Biol Res Commun 2017; 6(4):169-179
- The (-)-hyoscyamine, atropine and scopolamine (hyoscine) are three valuable tropane alkaloids while scopolamine is the most important member of this group for the pharmaceutical industry due to its h...
The (-)-hyoscyamine, atropine and scopolamine (hyoscine) are three valuable tropane alkaloids while scopolamine is the most important member of this group for the pharmaceutical industry due to its higher demand compared to hyoscyamine and atropine. Scopolamine is an anticholinergic reagent with several therapeutic applications. In the current study, the hairy roots culture of Daturametel was used as an advantageous method for production of scopolamine. The hairy roots are formed by Agrobacterium rhizogenes and have genetic stability, high growth rate and lateral branching. In this study, the effect of Bacillus cereus and Staphylococcus aureus as biotic elicitors on the production of scopolamine in D.metel hairy roots was investigated. The amount of scopolamine in the hairy roots was detected by HPLC analysis and compared with control samples after 0, 12 and 24 hours. Results showed that, B. cereus and S. aureus enhanced scopolamine production in the culture while the atropine content was decreased. Although in the control samples with no bacterial elicitation no scopolamine was detected, elicitation by B. cereus caused production of scopolamine and about 0.03 gram and 0.017 gram of it was detected in 100 gram dried D.metel hairy roots after 12 and 24 hours respectively. In S. aureus elicited hairy roots, scopolamine was not produced after 12 hours. However, about 0.025 gram of this tropane alkaloid was detected in 100 gram dried hairy roots after 24 hours. In conclusion, S. areus and B. cereus induced the scopolamine production in D. metel hairy roots.
- Comparison of two hyoscyamine 6β-hydroxylases in engineering scopolamine biosynthesis in root cultures of Scopolia lurida. [Journal Article]
- BBBiochem Biophys Res Commun 2018 Jan 31
- Scopolia lurida, a medicinal plant native to the Tibetan Plateau, is among the most effective producers of pharmaceutical tropane alkaloids (TAs). The hyoscyamine 6β-hydroxylase genes of Hyoscyamus n...
Scopolia lurida, a medicinal plant native to the Tibetan Plateau, is among the most effective producers of pharmaceutical tropane alkaloids (TAs). The hyoscyamine 6β-hydroxylase genes of Hyoscyamus niger (HnH6H) and S. lurida (SlH6H) were cloned and respectively overexpressed in hairy root cultures of S. lurida, to compare their effects on promoting the production of TAs, especially the high-value scopolamine. Root cultures with SlH6H/HnH6H overexpression were confirmed by PCR and real-time quantitative PCR, suggesting that the enzymatic steps defined by H6H were strongly elevated at the transcriptional level. Tropane alkaloids, including hyoscyamine, anisodamine and scopolamine, were analyzed by HPLC. Scopolamine and anisodamine contents were remarkably elevated in the root cultures overexpressing SlH6H/HnH6H, whereas that of hyoscyamine was more or less reduced, when compared with those of the control. These results also indicated that SlH6H and HnH6H promoted anisodamine production at similar levels in S. lurida root cultures. More importantly, HnH6H-overexpressing root cultures had more scopolamine in them that did SlH6H-overexpressing root cultures. This study not only provides a feasible way of overexpressing H6H to produce high-value scopolamine in engineered root cultures of S. lurida but also found that HnH6H was better than SlH6H for engineering scopolamine production.
- Determination of tropane alkaloids by heart cutting reversed phase - Strong cation exchange two dimensional liquid chromatography. [Journal Article]
- JCJ Chromatogr B Analyt Technol Biomed Life Sci 2018 Jan 01; 1072:70-77
- Current Chinese Pharmacopoeia (ChP) standards apply liquid extraction combined with one dimensional liquid chromatography (1DLC) method for determining alkaloids in herbal medicines. The complex pret...
Current Chinese Pharmacopoeia (ChP) standards apply liquid extraction combined with one dimensional liquid chromatography (1DLC) method for determining alkaloids in herbal medicines. The complex pretreatments lead to a low analytical efficiency and possible component loss. In this study, a heart cutting reversed phase - strong cation exchange two dimensional liquid chromatography (RP - SCX 2DLC) approach was optimized for simultaneously quantifying tropane alkaloids (anisodine, scopolamine and hyoscyamine) in herbal medicines and herbal medicine tablets without further treatment of the filtered extract. The chromatographic conditions were systematically optimized in terms of column type, mobile phase composition and flow rate. To improve peak capacity and obtain symmetric peak shape of alkaloids, a polar group embedded C18 column combined with chaotropic salts was used in the first dimension. To remove the disturbance of non-alkaloids, achieve unique selectivity and acquire symmetric peak shape of alkaloids, an SCX column combined with phosphate buffer was used in the second dimension. Method validation was performed in terms of linearity, precision (0.54-0.82%), recovery (94.1-105.2%), limit of detection (LOD) and limit of quantification (LOQ) of the three analytes varied between 0.067-0.115mgL-1and 0.195-0.268mgL-1, respectively. The method demonstrated superiority over 1DLC method in respect of resolution (less alkaloid co-eluted), sample preparation (no pretreatment procedure) and transfer rate (minimum component loss). The optimized RP - SCX 2DLC approach was subsequently applied to quantify target alkaloids in five herbal medicines and herbal medicine tablets from three different manufactures. The results demonstrated that the developed heart cutting RP - SCX 2DLC approach represented a new, strategically significant methodology for the quality evaluation of tropane alkaloid in related herbal medicines that involve complex chemical matrix.
- Localization and Organization of Scopolamine Biosynthesis in Duboisia myoporoides R. Br. [Journal Article]
- PCPlant Cell Physiol 2018 Jan 01; 59(1):107-118
- Tropane alkaloids (TAs), especially hyoscyamine and scopolamine, are important precursors for anticholinergic and antispasmodic drugs. Hyoscyamine and scopolamine are currently obtained at commercial...
Tropane alkaloids (TAs), especially hyoscyamine and scopolamine, are important precursors for anticholinergic and antispasmodic drugs. Hyoscyamine and scopolamine are currently obtained at commercial scale from hybrid crosses of Duboisia myoporoides × Duboisia leichhardtii plants. In this study, we present a global investigation of the localization and organization of TA biosynthesis in a Duboisia myoporoides R. Br. wild-type line. The tissue-specific spatial distribution of TAs within D. myoporoides is presented, including quantification of the TAs littorine, 6-hydroxy hyoscyamine, hyoscyamine, scopolamine and, additionally, hyoscyamine aldehyde as well as scopolamine glucoside. Scopolamine (14.77 ± 5.03 mg g-1), and to a lesser extent hyoscyamine (3.01 ± 1.54 mg g-1) as well as 6-hydroxy hyoscyamine (4.35 ± 1.18 mg g-1), are accumulated in leaves during plant development, with the highest concentration of total TAs detected in 6-month-old plants. Littorine, an early precursor in TA biosynthesis, was present only in the roots (0.46 ± 0.07 mg g-1). During development, the spatial distribution of all investigated alkaloids changed due to secondary growth in the roots. Transcripts of pmt, tr-I and cyp80f1 genes, involved in early stages of TA biosynthesis, were found to be most abundant in the roots. In contrast, the transcript encoding hyoscyamine 6β-hydroxylase (h6h) was highest in the leaves of 3-month-old plants. This investigation presents the spatial distribution of biochemical components as well as gene expression profiles of genetic factors known to participate in TA biosynthesis in D. myoporoides. The results of this investigation may aid in future breeding or genetic enhancement strategies aimed at increasing the yields of TAs in these medicinally valuable plant species.
- Enhancing Tropane Alkaloid Production Based on the Functional Identification of Tropine-Forming Reductase in Scopolia lurida, a Tibetan Medicinal Plant. [Journal Article]
- FPFront Plant Sci 2017; 8:1745
- Scopolia lurida, a native herbal plant species in Tibet, is one of the most effective producers of tropane alkaloids. However, the tropane alkaloid biosynthesis in this plant species of interest has ...
Scopolia lurida, a native herbal plant species in Tibet, is one of the most effective producers of tropane alkaloids. However, the tropane alkaloid biosynthesis in this plant species of interest has yet to be studied at the molecular, biochemical, and biotechnological level. Here, we report on the isolation and characterization of a putative short chain dehydrogenase (SDR) gene. Sequence analysis showed that SlTRI belonged to the SDR family. Phylogenetic analysis revealed that SlTRI was clustered with the tropine-forming reductases. SlTRI and the other TA-biosynthesis genes, including putrescine N-methyltransferase (SlPMT) and hyoscyamine 6β-hydroxylase (SlH6H), were preferably or exclusively expressed in the S. lurida roots. The tissue profile of SlTRI suggested that this gene might be involved in tropane alkaloid biosynthesis. By using GC-MS, SlTRI was shown to catalyze the tropinone reduction to yield tropine, the key intermediate of tropane alkaloids. With the purified recombinant SlTRI from Escherichiacoli, an enzymatic assay was carried out; its result indicated that SlTRI was a tropine-forming reductase. Finally, the role of SlTRI in promoting the tropane alkaloid biosynthesis was confirmed through metabolic engineering in S. lurida. Specifically, hairy root cultures of S. lurida were established to investigate the effects of SlTRI overexpression on tropane alkaloid accumulation. In the SlTRI-overexpressing root cultures, the hyoscyamine contents were 1.7- to 2.9-fold higher than those in control while their corresponding scopolamine contents were likewise elevated. In summary, this functional identification of SlTRI has provided for a better understanding of tropane alkaloid biosynthesis. It also provides a candidate gene for enhancing tropane alkaloid biosynthesis in S. lurida via metabolic engineering.
- Novel Microbial Sources of Tropane Alkaloids: First Report of Production by Endophytic Fungi Isolated from Datura metel L. [Journal Article]
- CMCurr Microbiol 2018; 75(2):206-212
- Eighteen endophytic fungi were isolated from various tissues of Datura metel and genes encoding for putrescine N-methyltransferase (PMT), tropinone reductase 1 (TR1) and hyoscyamine 6β-hydroxylase (H...
Eighteen endophytic fungi were isolated from various tissues of Datura metel and genes encoding for putrescine N-methyltransferase (PMT), tropinone reductase 1 (TR1) and hyoscyamine 6β-hydroxylase (H6H) were used as molecular markers for PCR-based screening approach for tropane alkaloids (TAs) producing endophytic fungi. These fungi were identified taxonomically by sequence analysis of the internal transcribed spacer region (ITS1-5.8S-ITS2) and also based on morphological characteristics of the fungal spore as Colletotrichum boninense, Phomopsis sp., Fusarium solani, Colletotrichum incarnatum, Colletotrichum siamense and Colletotrichum gloeosporioides. The production of TAs hyoscyamine and scopolamine by the fungi has been ascertained using chromatography and spectroscopy methods by comparison with the standards. Among the fungi, the highest yields of hyoscyamine (3.9 mg/L) and scopolamine (4.1 mg/L) were found in C. incarnatum culture. This is the first report of endophytic fungi possess the PMT, TR1 and H6H genes and produces TAs. These endophytic fungi have significant potential to be applied in fermentation technology to meet the demands for TAs economically.
- Acute poisoning due to ingestion of Datura stramonium - a case report. [Journal Article]
- RJRom J Anaesth Intensive Care 2017; 24(1):65-68
- Datura stramonium (DS) is a widespread annual plant, containing atropine, hyoscyamine, and scopolamine, which can produce poisoning with a severe anticholinergic syndrome. Teenagers ingest the roots,...
Datura stramonium (DS) is a widespread annual plant, containing atropine, hyoscyamine, and scopolamine, which can produce poisoning with a severe anticholinergic syndrome. Teenagers ingest the roots, seeds or the entire plant to obtain its hallucinogenic and euphoric effects. We presented the case of a 22 year old male who was admitted to the Emergency Room in a coma after consuming Datura stramonium, 2 hours earlier. The patient presented with fever, tachycardia with right bundle branch block, and urinary retention. Rapid sequence induction and intubation was performed immediately, with sedation and assisted-control mechanical ventilation, after being transferred to the Intensive Care Unit. The patient received activated charcoal, in repeated doses, external and internal cooling was applied, and an infusion of neostigmine was started. The biological assessment revealed rhabdomyolysis and prevention of renal failure was initiated. After a proper neurological evaluation, 36 hours after using Datura stramonium, the patient was extubated and transferred to the Psychiatric ward for further assessment and care.
- Genetically engineered hairy root cultures of Hyoscyamus senecionis and H. muticus: ploidy as a promising parameter in the metabolic engineering of tropane alkaloids. [Journal Article]
- PCPlant Cell Rep 2017; 36(10):1615-1626
- 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...
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. 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.
- Pyridostigmine Induced Prolonged Asystole in a Patient with Myasthenia Gravis Successfully Treated with Hyoscyamine. [Journal Article]
- CRCase Rep Cardiol 2017; 2017:6956298
- Reversible acetylcholinesterase inhibitors are used as first-line treatment for myasthenia gravis. They improve symptoms by increasing concentration of acetylcholine at the neuromuscular junction and...
Reversible acetylcholinesterase inhibitors are used as first-line treatment for myasthenia gravis. They improve symptoms by increasing concentration of acetylcholine at the neuromuscular junction and stimulating nicotinic receptors. Serious bradyarrhythmias can occur from muscarinic stimulation in heart, which in rare cases may progress to asystole. These patients can initially be managed with hyoscyamine, a muscarinic antagonist. Persistence of bradyarrhythmias even after hyoscyamine treatment may warrant pacemaker placement. We present a case of 65-year-old female patient who presented with diplopia, dysphagia, and muscle weakness who was diagnosed with myasthenia gravis. She developed significant sinoatrial node block with prolonged asystole after starting treatment with pyridostigmine which was successfully treated with hyoscyamine, thus avoiding pacemaker placement.
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- Enhanced production of hyoscyamine and scopolamine from genetically transformed root culture ofHyoscyamus reticulatusL. elicited by iron oxide nanoparticles. [Journal Article]
- VCIn Vitro Cell Dev Biol Plant 2017; 53(2):104-111
- The medicinal plantHyoscyamus reticulatusL. is a rich source of hyoscyamine and scopolamine, the tropane alkaloids. The use of hairy root cultures has focused significant attention on production of i...
The medicinal plantHyoscyamus reticulatusL. 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 withAgrobacterium rhizogenesand 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-1FeNPs. 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-1FeNPs 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.