Tropane Alkaloid Content Research Articles

A novel bHLH gene responsive to low nitrogen positively regulates the biosynthesis of medicinal tropane alkaloids in Atropa belladonna

Medicinal tropane alkaloids (TAs), including hyoscyamine, anisodamine and scopolamine, are essential anticholinergic drugs specifically produced in several solanaceous plants. Atropa belladonna is one of the most important medicinal plants that produces TAs. Therefore, it is necessary to cultivate new A. belladonna germplasm with the high content of TAs. Here, we found that the levels of TAs were elevated under low nitrogen (LN) condition, and identified a LN-responsive bHLH transcription factor (TF) of A. belladonna (named LNIR) regulating the biosynthesis of TAs. The expression level of LNIR was highest in secondary roots where TAs are synthesized specifically, and was significantly induced by LN. Further research revealed that LNIR directly activated the transcription of hyoscyamine 6β-hydroxylase gene (H6H) by binding to its promoter, which converts hyoscyamine into anisodamine and subsequently epoxidizes anisodamine to form scopolamine. Overexpression of LNIR upregulated the expression levels of TA biosynthesis genes and consequently led to the increased production of TAs. In summary, we functionally identified a LN-responsive bHLH gene that facilitated the development of A. belladonna with high-yield TAs under the decreased usage of nitrogen fertilizer.

Potential Application of Invasive Plant Species Datura innoxia for the Scopolamine Extracts of the Plant Organs and Analysis Using UV–VIS Spectrophotometry

Downy thorn-apple Datura innoxia (Solanaceae) is an invasive plant species which can be introduced either accidentally or deliberately and have the ability to acclimatize in new environmental conditions. Scopolamine is a natural alkaloid which occur in several Datura species, including Datura innoxia. Occurrence of alkaloid in all plant organs is crucially important from the viewpoint of medical use, but also as a risk of toxicity for humans and animals, this paper presents the influence of alcoholic solvents on the separation ability of tropane alkaloids using a conventional extraction method (Soxhlet extraction) in order to investigate the content of scopolamine from Datura innoxia dry biomass from Romania at the maturity stage. Two solvents were selected, investigated and compared, including ethanol (96%) and 1-butanol (99.6%). The results showed that 1-butanol was most suitable for the extraction of scopolamine from Datura innoxia areal parts (leaves, flowers, seeds, stem and root) then ethanol because of the high degree of lipophilicity of this alkaloid. The quantitative analysis was performed using UV–VIS spectrophotometry technique. The calibration curve for the analyte under the optimum conditions was obtained with a proper correlation coefficient of 0.9930. Scopolamine was identified in all vegetative organs with higher concentration in 1-butanol and the total content (µg g−1) was 186.87 in leaves, 150.89 in flowers, 63.27 in seeds, 42.50 in stem and 58.10 in root. These studies provide new insights into the potential use of invasive plant species Datura innoxia for extraction of the content of tropane alkaloids, especially scopolamine using different solvents regarding the toxicity and therapeutic potential of this plant alkaloid.

Issues of Safety of Herb Mixes and Food Supplements Contaminated with Tropane Alkaloids: A Review

Background: Nutritional supplements, herbal assemblages, and herbal teas are of particular interest in the global market of specialized food products, with the issues of a safe use becoming particularly relevant in the light of the reports on their potential contamination with tropane alkaloids, which content ranges from 0.01 % to 3 % depending on the type of raw materials. Objective: Analysis of the safety of dietary supplements, herbs and herbal teas contaminated with tropane alkaloids, based on data currently presented in domestic and international scientific publications. Materials and methods: We reviewed and analyzed literature sources found in Scopus, PubMed and RSCI databases for tropane alkaloids. The initial sample included 49 articles, of which 19 articles were excluded following a primary analysis. The selection criteria included the presence of classification of tropane alkaloids, their contents in herbs and foods, methods of detection, and potential human health effects. We selected 30 full-text publications meeting the above criteria. The study results were systematized by the type of intervention. Results: The analysis showed that the content of tropane alkaloids in some herbal teas by the sum of atropine and scopolamine can exceed the established level of a single safe intake (0.016 µg/kg body weight). Since scopolamine is a derivative of hyoscyamine (atropine), safety of foods and raw materials should be assessed by their content of atropine that will allow effective monitoring of contamination with tropane alkaloids of all types of the specified food products for the purpose of their safe use. Conclusion: For the safe use of herbal preparations and plant-based dietary supplements, it is recommended to monitor these types of food products for the presence of tropane alkaloids in them. It is advisable to develop a method for their analysis using high-performance liquid chromatography coupled with mass spectrometry.

A preliminary pharmacophylogenetic study of Solanaceae medicinal plants containing tropane alkaloids

The Solanaceae plants distributed in China belong to 105 species and 35 varietas of 24 genera. Some medicinal plants of Solanaceae are rich in tropane alkaloids(TAs), which have significant pharmacological activities. In this paper, the geographical distribution, chemical components, traditional therapeutic effect, pharmacological activities, and biosynthetic pathways of TAs in Solanaceous plants were summarized. Besides, the phylogeny of medicinal plants belonging to Solanaceae was visualized by network diagram. Fourteen genera of Solanaceae plants in China contain TAs and have medical records. TAs mainly exist in Datura, Anisodus, Atropa, Physochlaina, and Hyoscyamus. The TAs-containing species were mainly concentrated in Southwest China, and the content of TAs was closely related to plant distribution area and altitude. The Solanaceae plants containing TAs mainly have antispasmodic, analgesic, antiasthmatic, and antitussive effects. Modern pharmacological studies have proved the central sedative, pupil dilating, glandular secretion-inhibiting, and anti-asthma activities of TAs. These pharmacological activities provide a reasonable explanation for the traditional therapeutic efficacy of tropane drugs. In this paper, the geographical distribution, chemical components, traditional therapeutic effect, and modern pharmacological activities of TAs-containing species in Solanaceae were analyzed for the first time. Based on these data, the genetic relationship of TAs-containing Solanaceae species was preliminarily discussed, which provided a scientific basis for the basic research on TAs-containing solanaceous species and was of great significance for the development of natural medicinal plant resources containing TAs.

Tropane alkaloid biosynthesis in Datura innoxia Mill. roots and their differential transport to shoots

Pharmacologically important tropane alkaloids such as hyoscyamine, and scopolamine, are synthesized in the roots of several Solanaceae and also accumulate in their shoots. The tropane alkaloids content in roots of D. innoxia seedlings was almost 3-fold higher than in their shoots. Moreover, scopolamine accounted for 77 % of the total tropane alkaloid content in shoots as compared to only 20 % in roots. All candidate genes in the scopolamine biosynthetic pathway were significantly expressed in D. innoxia mature roots with limited expression in mature leaves. Using exogenous isotopically labeled 2H5 L-phenylalanine, we show here that root tissues, but not detached shoots, readily biosynthesize scopolamine. Tropane alkaloids newly synthesized by roots, and preferentially scopolamine, were further transported to shoots. Our evidence indicates that D. innoxia seedlings have the ability to synthesize tropane alkaloids in their roots and differentially transport them to the shoots.

Comparison of Profiling of Hairy Root of Two Tibetan Medicinal Plants Przewalskia tangutica Maxim. and Anisodus tanguticus Maxim.

Tropane Alkaloids (TAs) are important drugs for curing many diseases in the medical industry. To sustainably exploit TA resources in endangered traditional Tibetan herbs, the hairy root (HR) systems of Przewalskia tangutica Maxim. and Anisodus tanguticus Maxim. were compared under the same culture conditions. The results indicated that both the Agrobacterium rhizogenes strains and explants affected the HR induction frequency, MSU440, A4 and LBA9402 strains could induce hairy roots following infection of cotyledon and hypocotyl of A. tanguticus while LBA9402 could not induce HR on either explants of P. tangutica. The efficiency of LBA9402 was higher than A4 and MSU440 on A. tanguticus and A4 was better strain than MSU440 on P. tangutica. The hypocotyl explant was more suitable for P.tangutica and cotyledon explant was better for A.tangutica with a transformation frequency of 33.3% (P. tangutica) and 82.5% (A. tanguticus), respectively. In a flask reactor system, both the growth curves of HR for two species both appeared to be "S" curve; however, the HR of P. tangutica grew more rapidly than that of A. tanguticus, and the latter accumulated more biomass than the former. As the culture volume increased, the HR proliferation coefficient of both the species increased. HPLC analysis results showed that the content of TAs in the HR of P. tangutica was 257.24mg/100g·DW, which was more than that of A. tanguticus HR (251.08mg/100g·DW), and the anisodamine in the Pt- HR was significantly higher than that in At-HR. Moreover, tropane alkaloids in the HR of the two species were all significantly higher than that of the roots of aseptic seedlings. Our results suggest that HR of P. tangutica and A. tanguticus both could provide a useful platform for sustainable utilization of two Tibetan medicinal plants in the Qinghai-Tibetan Plateau in the future.

On the regulate tropane alkaloids in grain products

Tropane alkaloids (TA) are metabolites of various plant species in the families Brassicaceae, Solanaceae and Erythroxylaceae. Seeds of these weeds are found in flax, soy, sorghum, millet, sunflower, buckwheat and food products obtained from them, in herbals used as a part of food supplements and herbal teas. Despite the fact that the contamination of grain seeds by various parts of weeds reduces by sorting and clean up, a certain amount of TA gets into processed foods. An analysis of the consumption of TA with all types of foods, conducted by the European food safety Agency (EFSA), showed that TA intake with ration of various population groups can exceed the established level of single safe intake (ARfD) - 0.016 μg/kg of body weight (by 11-18% for adults, and by 5-25% for children depending on the age). The aim of the research was to justify the need to introduce regulations for the safe content of TA in grain products. Material and methods. The study process used hygiene, analytical methods, expert assessments on the basis of generalization and analysis of contemporary scientific researches published in databases Scopus, Web of Science, PubMed, RISC, Russian and international regulatory and legislative documents. Results. The established risks associated with TA contamination of grain and grain-based foods, as well as food supplements based on herbs and herbal teas, make it necessary to monitor food products for it's presence. Conclusion. Due to the greater study of the risks associated with the presence of atropine and scopolamine in foods, scientific justification of safe levels of these TA in foods based on cereals, including baby foods for children under 3 years old containi ng millet, sorghum, buckwheat or products based on these cereals is required.

Erythroxylum in Focus: An Interdisciplinary Review of an Overlooked Genus.

The genus Erythroxylum contains species used by indigenous people of South America long before the domestication of plants. Two species, E. coca and E. novogranatense, have been utilized for thousands of years specifically for their tropane alkaloid content. While abuse of the narcotic cocaine has impacted society on many levels, these species and their wild relatives contain untapped resources for the benefit of mankind in the form of foods, pharmaceuticals, phytotherapeutic products, and other high-value plant-derived metabolites. In this review, we describe the current state of knowledge of members within the genus and the recent advances in the realm of molecular biology and biochemistry.

Alkaloid chemodiversity in Mandragora spp. is associated with loss-of-functionality of MoH6H, a hyoscyamine 6β-hydroxylase gene

Mandrakes (Mandragora spp., Solanaceae) are known to contain tropane alkaloids and have been used since antiquity in traditional medicine. Tropane alkaloids such as scopolamine and hyoscyamine are used in modern medicine to treat pain, motion sickness, as eye pupil dilators and antidotes against organo-phosphate poisoning. Hyoscyamine is converted to 6β-hydroxyhyoscyamine (anisodamine) and scopolamine by hyoscyamine 6β-hydroxylase (H6H), a 2-oxoglutarate dependent dioxygenase. We describe here a marked chemo-diversity in the tropane alkaloid content in Mandragora spp. M. officinarum and M. turcomanica lack anisodamine and scopolamine but display up to 10 fold higher hyoscyamine levels as compared with M. autumnalis. Transcriptomic analyses revealed that H6H is highly conserved among scopolamine-producing Solanaceae. MoH6H present in M. officinarum differs in several amino acid residues including a homozygotic mutation in the substrate binding region of the protein and its prevalence among accessions was confirmed by Cleaved-Amplified-Polymorphic-Sequence analyses. Functional expression revealed that MaH6H, a gene isolated from M. autumnalis encodes an active H6H enzyme while the MoH6H sequence isolated from M. officinarum was functionally inactive. A single G to T mutation in nucleotide 663 of MoH6H is associated with the lack of anisodamine and scopolamine in M. officinalis.

Influence of nano-zinc oxide on tropane alkaloid production, h6h gene transcription and antioxidant enzyme activity in Hyoscyamus reticulatus L. hairy roots.

The use of nanotechnology and biotechnology to improve the production of plant bioactive compounds is growing. Hyoscyamus reticulatus L. is a major source of tropane alkaloids with a wide therapeutic use, including treatment of Parkinson's disease and to calm schizoid patients. In the present study, hairy roots were obtained from two-week-old cotyledon explants of H. reticulatus L. using the A7 strain of Agrobacterium rhizogenes. The effects of different concentrations of the signaling molecule nano-zinc oxide (ZnO) (0, 50, 100 and 200mg/L), with three exposure times (24, 48 and 72 h), on the growth rate, antioxidant enzyme activity, total phenol contents (TPC), tropane alkaloid contents and hyoscyamine-6-beta-hydroxylase (h6h) gene expression levels were investigated. Growth curve analysis revealed a decrease in fresh and dry weight of ZnO-treated hairy roots compared to the control. ANOVA results showed that the antioxidant activity of the enzymes catalase, guaiacol peroxidase and ascorbate peroxidase was significantly higher in the ZnO-treated hairy roots than in the control, as was the TPC. The highest levels of hyoscyamine (37%) and scopolamine (37.63%) were obtained in hairy roots treated with 100mg/L of ZnO after 48 and 72 h, respectively. Semi-quantitative RT-PCR analysis revealed the highest h6h gene expression was in hairy roots treated with 100mg/L of ZnO after 24 h. It can be concluded that ZnO is as an effective elicitor of tropane alkaloids such as hyoscyamine and scopolamine due to its enhancing effect on expression levels of the biosynthetic h6h gene.

Pseudomonas spp. increases root biomass and tropane alkaloid yields in transgenic hairy roots of Datura spp.

Transgenic hairy roots of Datura spp., established using strain A4 of Agrobacterium rhizogenes, are genetically stable and produce high levels of tropane alkaloids. To increase biomass and tropane alkaloid content of this plant tissue, four Pseudomonas strains, Pseudomonas fluorescens P64, P66, C7R12, and Pseudomonas putida PP01 were assayed as biotic elicitors on transgenic hairy roots of Datura stramonium, Datura tatula, and Datura innoxia. Alkaloids were extracted from dried biomass, and hyoscyamine and scopolamine were quantified using liquid chromatography-tandem mass spectrometry analysis. D. stramonium and D. innoxia biomass production was stimulated by all Pseudomonas spp. strains after a 5-d treatment. All strains of P. fluorescens increased hyoscyamine yields compared to untreated cultures after both 5 and 10 d of treatment. Hyoscyamine yields were highest in D. tatula cultures exposed to a 5-d treatment with C7R12 (16.633 + 0.456 mg g−1 dry weight, a 431% increase) although the highest yield increases compared to the control were observed in D. stramonium cultures exposed to strains P64 (511% increase) and C7R12 (583% increase) for 10 d. D. innoxia showed the highest scopolamine yields after elicitation with P. fluorescens strains P64 for 5 d (0.653 + 0.021 mg g−1 dry weight, a 265% increase) and P66 for 5 and 10 d (5 d, 0.754 + 0.0.031 mg g−1 dry weight, a 321% increase; 10 d 0.634 + 0.046 mg g−1 dry weight, a 277% increase). These results show that the Pseudomonas strains studied here can positively and significantly affect biomass and the yields of hyoscyamine and scopolamine from transgenic roots of the three Datura species.

Production of tropan alkaloids in the in vitro and callus cultures of Hyoscyamus aureus and their genetic stability assessment using ISSR markers.

Green wild plants (dirctly before flowering) and seeds of Hyoscyamus aureus were collected from natural habitat at Al Qalamon region in Syria. Seeds were surface sterilized and cultured in vitro, after 21 days from germination stem-derived callus was induced on two different nutrient media. Tropane alkaloids were extracted from wild plants and 30 days old in vitro plants and callus, and then analyzed using GC-MS. Genetic variation was also studied between the wild and in vitro plants and the callus culture lines using twenty ISSR markers. The results showed that there were significant variations in tropane alkaloids contents between the wild plants, the in vitro plants and the callus culture lines. The highest content of hyoscyamine was in callus on line A medium, but the highest content of scopolamine was in the wild plants. However, the lowest content of tropane alkaloids was in callus on line B medium. Also the ISSR analyses showed that there was genetic variation between the wild and in vitro plants and the callus culture lines.

Changes of Ultrastructure of Cells and Tropane Alkaloid Contents during Organogenesis of Scopolia parviflora Callus Cultures

Changes of Ultrastructure of Cells and Tropane Alkaloid Contents during Organogenesis of Scopolia parviflora Callus Cultures

A small-scale proteomic approach reveals a survival strategy, including a reduction in alkaloid biosynthesis, in Hyoscyamus albus roots subjected to iron deficiency.

Hyoscyamus albus is a well-known source of the tropane alkaloids, hyoscyamine and scopolamine, which are biosynthesized in the roots. To assess the major biochemical adaptations that occur in the roots of this plant in response to iron deficiency, we used a small-scale proteomic approach in which 100 mg of root tips were treated with and without Fe, respectively, for 5 days. Two-dimensional mini gels showed that 48 spots were differentially accumulated between the two conditions of Fe availability and a further 36 proteins were identified from these spots using MALDI-QIT-TOF mass spectrometry. The proteins that showed elevated levels in the roots lacking Fe were found to be associated variously with carbohydrate metabolism, cell differentiation, secondary metabolism, and oxidative defense. Most of the proteins involved in carbohydrate metabolism were increased in abundance, but mitochondrial NAD-dependent malate dehydrogenase was decreased, possibly resulting in malate secretion. Otherwise, all the proteins showing diminished levels in the roots were identified as either Fe-containing or ATP-requiring. For example, a significant decrease was observed in the levels of hyoscyamine 6β-hydroxylase (H6H), which requires Fe and is involved in the conversion of hyoscyamine to scopolamine. To investigate the effects of Fe deficiency on alkaloid biosynthesis, gene expression studies were undertaken both for H6H and for another Fe-dependent protein, Cyp80F1, which is involved in the final stage of hyoscyamine biosynthesis. In addition, tropane alkaloid contents were determined. Reduced gene expression was observed in the case of both of these proteins and was accompanied by a decrease in the content of both hyoscyamine and scopolamine. Finally, we have discussed energetic and Fe-conservation strategies that might be adopted by the roots of H. albus to maintain iron homeostasis under Fe-limiting conditions.

Tropane alkaloids of Atropa belladonna L.: in vitro production and pharmacological profile

ObjectiveThe aim of the present work was to study the production of tropane alkaloids by in vitro cultures of Atropa belladonna L. and to evaluate the anticonvulsant, antinociceptive, motor incoordination, and antioxidant activities of both in vitro and original plant extracts.BackgroundA. belladonna is a very important medicinal plant with multipurpose therapeutic effects. The yield of its alkaloid content is very low, which makes it difficult for industrial application.Materials and methodsMurashige and Skoog media were used for callus and plant differentiation induction from leaf explants of A. belladonna L. Qualitative and quantitative analysis of alkaloids was carried out using high-performance liquid chromatography. The anticonvulsant activity was screened by the pentylenetetrazole seizure test. The antinociceptive activity was evaluated by adopting the writhing test, whereas motor incoordination was evaluated using the rotarod test. In addition, antioxidant activity was estimated using the 2,2'-diphenyl-1-picrylhydrazyl radical-scavenging test.ResultsCallus and differentiated plants were successfully induced in Murashige and Skoog media supplemented with growth regulators. High-performance liquid chromatography analysis revealed the production of higher concentrations of tropane alkaloids in differentiated plants than in the original plant. Anticonvulsant and antinociceptive activities, motor incoordination, and the antioxidant effect of callus extracts were much higher than those of the original plant leaf extract.ConclusionPlant tissue culture could be considered as an efficient and alternative source of continuous supply of tropane alkaloids with potent anticonvulsant, antinociceptive, motor incoordination, and antioxidant activities. It is also a powerful tool for producing A. belladonna strain with a high tropane alkaloid content.

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

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

Tropane alkaloids of Atropa belladonna L. and Atropa acuminata Royle ex Miers plants

The purpose of the present study was to determine the tropane alkaloid content in different parts of the wild and cultivated Atropa belladonna L. and wild Atropa acuminata Royle ex Miers in Iran. Determination of alkaloids was performed by high-performance liquid chromatography (HPLC) method. Samples were extracted with chloroform- methanol- cc. ammonia 15:15:1(v/v/v). HPLC separation was performed on two C8 columns. An isocratic mobile phase of acetonitrile- 50 mM phosphate buffer (pH 2.95) 10:90 and 20:80(v/v) was used. Peaks were identified by standards and diode-array detection. Scopolamine and atropine were determined by external method at 210 nm. The total alkaloid content of the A. belladonna L. of sari-kiassar road was determined by a HPLC method to be 2.88% for the leaves and 8.06% for the roots and 1.42% for the stem. The total alkaloid content of the cultivated A. belladonna L. was 1.76% for the leaves, 3.3% for the roots, 1.42% for the stem and 4.82% for the seeds. Total alkaloid content of the leaves of A. acuminata was 5.88%. In all samples the percentage of atropine was higher than scopolamine. The results showed that A. belladonna and A. acuminata constitutes the major storage site and had the highest amount of these alkaloids. These alkaloids may be transported to other plant organs providing potential deterrent and antimicrobial activities. Key words: Atropa belladonna L., Atropa acuminata Royle ex Miers, high-performance liquid chromatography (HPLC), Tropane alkaloids.

Simultaneous determination of atropine and scopolamine in different parts of Hyoscyamus arachnoideus Pojark plants by high-performance liquid chromatography (HPLC)

A reverse phase high-performance liquid chromatography (HPLC) equipped with UV-PDA detector was used for the analysis of main tropane alkaloids, scopolamine and atropine, in the roots, aerial parts and seeds of Hyoscyamus arachnoideus Pojark plants collected from five different parts of Iran. Results showed that scopolamine was only the main alkaloid of two H. arachnoideus populations seeds, while atropine was the major alkaloid of almost all parts, especially roots. The highest and lowest content of tropane alkaloids was observed in populations from Zangane village and Ashtian Mountain, respectively. The alkaloid content of seeds was in general more than other parts tested. The linearity of the method was in the range of 4 to 400 µg/ml for atropine and 0.8 to 80 µg/ml for scopolamine. Limit of detection (LOD) and limit of quantification (LOQ) values were 5.15 and 17.4 µg/ml for atropine and 1.92 and 6.4 µg/ml for scopolamine.

Effects of different elicitors on yield of tropane alkaloids in hairy roots of Anisodus acutangulus

The four tropane alkaloids have played a pivotal role in controlling diseases such as the toxic and septic shock, the organophosphorus poison and the acute lung injury. Here, the elicitation effect of different elicitors on the production of tropane alkaloids and the molecular mechanism of enzyme genes in the pathway was firstly demonstrated in hairy roots of Anisodus acutangulus. The results showed ethanol, methyl jasmonate and Ag(+) could improve the accumulation of tropane alkaloids up to 1.51, 1.13 and 1.08 times after 24 h treatment, respectively (P < 0.05), whereas salicylic acid decreased the average content of tropane alkaloids. Furthermore, expression profile analysis results revealed that up-regulation of hyoscyamine-6b-hydroxylase (AaH6H) and little regulation of tropinone reducase II (AaTR2) elicited by ethanol, increased expression of putrescine N-methyltransferase I (AaPMT1) elicited by Ag(+), elevated expression of tropinone reducase I (AaTR1) elicited by methyl jasmonate, respectively, resulted in tropane alkaloids improvement. Our results showed that hairy root culture of A. acutangulus in combination with elicitors was a promising way for production of tropane alkaloids in the future.

Tropane alkaloid profiling of hydroponic Datura innoxia mill. Plants inoculated with Agrobacterium rhizogenes

Hydroponics has been shown as a possible way to produce high quality plant biomass with improved phytochemical levels. Nevertheless, effects of plant biotic and abiotic environment can lead to drastic changes and plant growth conditions must be optimised. To evaluate how much microbes and Agrobacterium rhizogenes TR7 wild strain may affect the tropane alkaloid profile in Datura innoxia Mill. plants cultivated in hydroponic conditions. Datura innoxia Mill. plants were cultivated in hydroponic with sterile or non-sterile conditions. For half of the non-sterile plants, Agrobacterium rhizogenes TR7 strain was added to the nutrient solution for hydroponics. The tropane alkaloid content of leaves and roots was analysed by UFLC/ESI-HRMS and MS/MS. The metabolite profiles were compared using partial least square-discriminant analysis. In sterile conditions, aerial parts contained more scopolamine than the roots. However, the diversity of tropane alkaloids was greater in roots. Furthermore, 21 known compounds and four non-elucidated tropane alkaloids were found. The tropane alkaloid profile was shown to be statistically different between sterile and non-sterile hydroponic conditions. The levels of 3-acetoxy-6-hydroxytropane and 3-hydroxylittorine were higher in plants inoculated with A. rhizogenes. Five other tropane compounds were found in higher amounts in non-axenic control plants. Hyoscyamine and scopolamine total contents were much higher in the whole plant co-cultivated with A. rhizogenes TR7 than in controls. Furthermore, the leaves and roots of axenic plants contained more alkaloids than non-sterile ones. In hydroponic conditions, microbes induced variations of the phytochemical levels. Addition of A. rhizogenes TR7 into the nutrient solutions improved the total hyoscyamine and scopolamine production.