Cloning and expression of the key enzyme hyoscyamine 6 beta-hydroxylase gene (DaH6H) in scopolamine biosynthesis of Datura arborea / 药学学报

Hyoscyamine 6 beta-hydroxylase (H6H) is the last rate-limiting enzyme directly catalyzing the formation of scopolamine in tropane alkaloids (TAs) biosynthesis pathway. It is the primary target gene in the genetic modification of TAs metabolic pathway. Full-length cDNA and gDNA sequences of a novel H6H gene were cloned from Datura arborea (DaH6H, GenBank accession numbers for cDNA and gDNA are KR006981 and KR006983, respectively). Nucleotide sequence analysis reveals an open reading frame of 1375 bp encoding 347 amino acids in the cDNA of DaH6H, while the gDNA of DaH6H contains four exons and three introns, with the highest similarity to the gDNA of H6H from D. stramonium. DaH6H also exhibited the most identity of 90.5% with DsH6H in amino acids and harbored conserved 2-oxoglutarate binding motif and two iron binding motifs. The expression level of DaH6H was highest in the mature leaf, followed by the secondary root, and with no expression in the primary root based on qPCR analysis. Its expression was inhibited by MeJA. DaH6H was expressed in E. coli and a 39 kD recombinant protein was detected in SDS-PAGE. Comparison of the contents of scopolamine and hyoscyamine in various TAs-producing plants revealed that D. arborea was one of the rare scopolamine predominant plants. Cloning of DaH6H gene will allow more research in the molecular regulatory mechanism of TAs biosynthesis in distinct plants and provide a new candidate gene for scopolamine metabolic engineering.

Effect of Flos Daturae Alkaloids on TGF-beta1-induced Epithelial-Mesenchymal Transition of Human Pulmonary Adenocarcinoma A549 Cells / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the effect of Flos Daturae alkaloids (FDA) on TGF-beta1-1uuuu;U epithelial-mesenchymal transition (EMT) of human pulmonary adenocarcinoma A549 cells.</p><p><b>METHODS</b>A549 cells in vitro cultured were randomly divided into 5 groups, i.e., the blank control group, the TGF-beta1 group, the low dose FDA group, the medium dose FDA group, and the high dose FDA group. The morphologies of A549 cells were observed. Expressions of cytokeratin (CK)-19 and alpha-smooth muscle actin (alpha-SMA) were detected by Western blot and real-time PCR at 24, 48, and 72 h, respectively.</p><p><b>RESULTS</b>A549 cells in the TGF-beta1, group turned from cobblestone to spindle shape gradually. Those in low, medium and high dose FDA groups showed similar shapes to those of the TGF-beta1 group. There was no statistical difference in the morphology of A549 cells among the 3 dose FDA groups (P > 0.05). Western blot showed that, when compared with the blank control group, the expression of CK-19 was down-regulated, but the expression of alpha-SMA was up-regulated in the TGF-beta1 group (P < 0.01). Compared with the TGF-beta1, group, the expression of CK-19 was up-regulated, but the expression of alpha-SMA was suppressed in low, medium and high dose FDA groups (P < 0.01). The CK-19 expression obviously increased, but the alpha-SMA expression was suppressed in high dose FDA group at 72 h (P < 0.01). Real-time PCR results showed, as compared with the TGF-beta1 group, the mRNA expression of CK-19 was increased, but the mRNA expression of alpha-SMA was reduced in low, medium and high dose FDA groups (P < 0.01).</p><p><b>CONCLUSIONS</b>FDA had no effect on EMT morphological changes of TGF-beta1 induced A549 cells. FDA could reverse characteristic markers of A549 cells during EMT to some extent, such as expressions of CK-19 and alpha-SMA. The expression of CK-19 (as the epithelium marker) increased and the expression of alpha-SMA (as the mesenchymal marker) was reduced. Besides, they were most obviously seen in the high dose FDA group at 72 h in a dose- and time-dependent manner.</p>

A review on the pharmacological and toxicological aspects of Datura stramonium L / 中西医结合学报

Datura stramonium L., a wild-growing plant of the Solanaceae family, is widely distributed and easily accessible. It contains a variety of toxic tropane alkaloids such as atropine, hyoscamine, and scopolamine. In Eastern medicine, especially in Ayurvedic medicine, D. stramonium has been used for curing various human ailments, including ulcers, wounds, inflammation, rheumatism and gout, sciatica, bruises and swellings, fever, asthma and bronchitis, and toothache. A few previous studies have reported on the pharmacological effects of D. stramonium; however, complete information regarding the pharmacology, toxicity, ethnobotany and phytochemistry remains unclear. Ethnomedicinally, the frequent recreational abuse of D. stramonium has resulted in toxic syndromes. D. stramonium, in the form of paste or solution to relieve the local pain, may not have a deleterious effect; however, oral and systemic administration may lead to severe anticholinergic symptoms. For this reason, it is very important for individuals, mainly young people, to be aware of the toxic nature and potential risks associated with the use of this plant. This comprehensive review of D. stramonium includes information on botany, phytochemistry, pharmacology, toxicology and ethnomedicinal uses.

Identification of Daturae flos and its adulterants based on DNA barcoding technique / 药学学报

To identify the original plant of Daturae Flos from its adulterants by DNA barcoding, the sequences of ITS2, psbA-trnH, matK, rbcL of four species including Datura metel, Darura innoxia, Darura stramonium and Brugmansia arborea were compared and analyzed. The PCR and sequencing success rate of the four regions (ITS2, psbA-trnH, matK, rbcL) was 100%, 90%, 100% and 85%, respectively. Sequences were assembled with CodonCode Aligner. K2P distances were calculated and NJ tree was performed by MEGA 4.1. Thirty SNPs were found among ITS2 sequences, and 33 insert/deletes were found among psbA-trnH intergenic regions. The interspecific K2P distance of ITS2 and psbA-trnH was obviously higher than that of the intraspecific one. As to matK and rbcL, there was no "Barcoding Gap" existing between inter- and intra-specific distances. The NJ trees of the four regions/combinations were built separately. Samples of Brugmansia arborea were clustered into one clade, and the other species of Datura L. formed another clade. The results showed that either ITS2 or psbA-trnH was useful to identify Daturae Flos from its adulterants.

Anticholinergic Toxicity Following Ingestion of Angel's Trumpet: A Case Report

Angel's trumpet is a poisonous shrub recently imported to Korea for ornamental PURPOSE. It contains tropane alkaloids and the ingestion of any part of the plant can cause serious anticholinergic toxicity. Not only accidental ingestion, intentional intake as hallucinogen is not uncommon in the United States and Europe. We report a woman who ingested angel's trumpet by mistake and showed anticholinergic syndrome. The potential public medical harm of this widespread plant should be reported in the media, along with the associated dangers from misuse as a hallucinogen.

Historical evidence for a pre-Columbian presence of Datura in the Old World and implications for a first millennium transfer from the New World.

Datura (Solanaceae)is a small genus of plants that,for long, was thought to occur naturally in both the New and Old Worlds. However, recent studies indicate that all species in the genus originated in the Americas. This finding has prompted the conclusion that no species of Datura could have been present in the Old World prior to its introduction there by Europeans in the early 16th century CE. Further, the textual evidence traditionally cited in support of a pre-Columbian Old World presence of Datura species is suggested to be due to the misreading of classical Greek and Arabic sources. As a result, botanists generally accept the opinion that Datura species were transferred into the Old World in the post-Columbian period. While the taxonomic and geographic evidence for a New World origin for all the Datura species appears to be well supported, the assertion that Datura species were not known in the Old World prior to the 16th century is based on a limited examination of the pre-Columbian non-Anglo sources.We draw on old Arabic and Indic texts and southern Indian iconographic representations to show that there is conclusive evidence for the pre-Columbian presence of at least one species of Datura in the Old World. Given the systematic evidence for a New World origin of the genus, the most plausible explanation for this presence is a relatively recent but pre-Columbian (probably first millennium CE) transfer of at least one Datura species, D. metel, into the Old World. Because D. metel is a domesticated species with a disjunct distribution,this might represent an instance of human-mediated transport from the New World to the Old World, as in the case of the sweet potato (Ipomoea batatas).

PEG-induced fusion of phosphatidylcholine-liposomes with protoplasts and post-fusion evaluation of plating efficiency and enrichment in plasmamembrane phosphatidylcholine of protoplasts in Datura innoxia Mill.

Liposomes entrapping fluorescein diacetate were fused with protoplasts of Datura innoxia Mill by employing polyethylene glycol (PEG) as the fusogen. Factors that influence liposome-protoplast fusion were optimized as a function of PEG-concentration and incubation duration, liposome composition and surface charge and liposome:protoplast ratio. Phosphatidylcholine-liposomes were found ideal for the objectives of the study. Fusion index based on per cent fluorescing protoplasts varied among the protoplast types. PEG-incubation duration in the fusion assay and growth ability of protoplasts to form microcalli subsequent to liposome-protoplast fusion was determined based on protoplast plating-efficiency. Plating efficiency of post-fusion protoplasts increased due to incorporation of liposome-phosphatidylcholine in the plasmamembrane of protoplasts. Results are discussed in relation to the application of liposome-protoplast fusion system in selective modification of plasmamembrane phospholipids of protoplasts.

Production of gastrodin through biotransformation of p-hydroxybenzaldehyde by cell suspension cultures of Datura tatula L / 生物工程学报

The conversion of exogenous p-hydroxybenzaldehyde to p-hydroxy-methyl-phenol-beta-D-glucoside (gastrodin) was studied by using cell suspension culture of Datura tatula L. The chemical structure of this synthesized gastrodin was identified based on the spectral analysis and chemical evidence. The conversion procedure of p-hydroxybenzaldehyde into gastrodin by D. tatula L. cell suspension cultures was established. The synthesized gastrodin (II) was isolated from the ferment liquor and identified by spectral analysis. At the same time, the p-hydroxybenzyl alcohol (I) converted through biotransformation of p-hydroxybenzaldehyde by cell suspension cultures of D. tatula L. was also isolated and identified. The efficiency of glucosylation of p-hydroxybenzaldehyde was remarkably enhanced by adding salicylic acid (0.1 mg/L) and keeping the lower pressure (0.001MPa) in 25L airlift loop bioreactor. The biotransformation of exogenous p-hydroxybenzaldehyde to gastrodin by cell suspension culture of D. tatula L. is a promising approach.

Determination of scopolamine and atropine in Flos Daturae by RP-HPLC / 中国中药杂志

<p><b>OBJECTIVE</b>To develop a quantitative analytical procedure of scopolamine and atropine in Flos Daturae using RP-HPLC.</p><p><b>METHOD</b>The two alkaloids were separated on a Hypersil BDS C18 column (4.6 mm x 250 mm, 5 microm) with a mobile phase of 0.02 mol x L(-1) sodium acetate buffer (containing 0.02% triethanolamine and the pH was adjusted to 6.0 with acetic acid)-methanol (60:40) and a detection wavelength of 215 nm. The flow rate was 1.0 mL x min(-1) and the column temperature was maintained at room temperature.</p><p><b>RESULT</b>The mean recovery was (99.6 +/- 1.8)% for scopolamine and (100.4 +/- 1.5)% for atropine.</p><p><b>CONCLUSION</b>This method was simple, accurate and sensitive.</p>

Datura Stramonium Intoxication: A report of 2 cases

Datura stramonium is an annual herb, growing wild in the most part of our country. All parts of the plant contain belladonna alkaloid. Ingestion of Datura stramonium and the resulting toxic symptoms have been already reported throughout the world, but not in Korea. Two cases of acute anticholinergic symptom due to Datura stramonium intoxication are presented. The diagnosis in the first case was suspected on the basis of clinical signs and symptoms, comfirmed by the appearance of similar symptoms and signs in the second, history of ingestion of small amount of stramonium liquor and identification of the plant specimen obtained. Both recovered spontaneously without neurologic sequalae.