Enhancement of tropane alkaloids biosynthesis in Atropa belladonna hariy root by overexpression of HnCYP82M3 and DsTRI genes / 药学学报

Tropane alkaloids (TAs) are a class of anticholinergic drugs widely used in clinical practice and mainly extracted from plant, among which Atopa belladonna is the main commercial drug source. It is of great industrial value to obtain TAs in large quantities by plant metabolic engineering. In TAs pathway, cytochrome oxidase CYP82M3 catalyze the synthesis of tropinone and then tropinone reductase I (TRI) compete with TRII for tropinone to form tropine leading to the TAs synthesis (drainage). In this study, based on the "increasing flow and drainage" metabolic engineering strategy, two genes, namely HnCYP82M3 and DsTRI from Hyoscyamus niger and Datura stramonium, respectively, were overexpressed in the hair roots of A. belladonna, with a view to promote the TAs accumulation. The HnCYP82M3 gene was cloned from the root of H. niger, and it encoded amino acid with 91.7% sequence identity with AbCYP82M3 from A. belladonna. Overexpression of HnCYP82M3 alone did not affect the content of TAs in hair roots of A. belladonna, indicating that CYP82M3 was not a key enzyme in TAs biosynthesis. Simultaneous overexpression of HnCYP82M3 and DsTRI greatly promoted the accumulation of the three TAs, and the contents of hyoscyamine, anisodamine and scopolamine were 4.97 times, 2.83 times and 2.19 times that of the control, respectively, and the increase amplitude was greater than that of single overexpression of DsTRI. This study showed that the "increasing flow and drainage" strategy of enzyme genes co-expression at branch points was a promising metabolic engineering method to effectively improve the biosynthesis of TAs in A. belladonna, and laid a theoretical and technical foundation for the large-scale industrial acquisition of TAs.

A preliminary pharmacophylogenetic study of Solanaceae medicinal plants containing tropane alkaloids / 中国中药杂志

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.

Nitrogen metabolism and secondary metabolism regulation of Atropa belladonna by exogenous NO under NaCl stress / 中国中药杂志

Atropa belladonna seedlings were used as experimental materials and cultivated by soil culture method. Different concentrations(0,0.05,0.1,0.2,0.5 mmol·L~(-1))of NO donor sodium nitroprusside(SNP) were sprayed on the leaves. The effects of different concentrations of SNP and different treatment time(4,8,12,16 d) on nitrogen metabolism, secondary metabolite content, precursor content of tropane alkaloid synthesis pathway and expression of key enzyme genes under 100 mmol·L~(-1) NaCl stress were studied. The results showed that with the prolongation of salt stress, the nitrogen metabolism and the accumulation of secondary metabolites of A. belladonna were inhibited to some extent. After treatment with different concentrations of exogenous SNP, the ammonium nitrogen content decreased dramatically, and the contents of nitrate nitrogen, free amino acid, soluble protein and the activities of key enzymes of nitrogen metabolism(NR, GS, GDH) were all greatly improved; the contents of precursor amino acids(ornithine, arginine) and polyamines(Put, Spd, Spm) in the secondary metabolic pathway have increased to varying degrees. The qRT-PCR analysis showed that exogenous SNP treatment can effectively promote the high expression of key enzyme genes PMT, TRⅠ and H6H in the secondary metabolic pathway of A. belladonna, and the production of hyoscyamine and scopolamine were increased notably. In summary, the application of appropriate concentration of SNP can effectively alleviate the inhibition of salt stress on the nitrogen metabolism and secondary metabolism of Atropa belladonna, and enhance its salt tolerance. Overall, 0.1 mmol·L~(-1) and 0.2 mmol·L~(-1) SNP treatment achieved the most remarkable effect.

Identification of Chemical Constituents in Caulis of Erycibe schmidtii by UPLC-Q-Exactive Focus-MS/MS / 中国实验方剂学杂志

Objective:To rapidly recognize and identify the chemical constituents in caulis of Erycibe schmidtii by ultra-high performance liquid chromatography coupled with Q-Exactive Focus mass spectrometry (UPLC-Q-Exactive Focus-MS/MS). Method:Taking 80% methanol extract of E. schmidtii caulis as the test solution, the chemical constituents in caulis of E. schmidtii were analyzed and identified. Thermo Accucure aQ C18 column (2.1 mm×150 mm, 2.6 μm) was used for chromatographic separation with the mobile phase of methanol (A)-0.1% formic acid solution (B) for gradient elution (0-12 min, 5%-25%A; 12-20 min, 25%-30%A; 20-28 min, 30%-38%A; 28-40 min, 38%-42%A). Positive and negative ion monitoring modes and heated electrospray ion source (HESI) were used for mass spectrographic analysis. The scanning range was m/z 80-1 200. Result:A total of 42 chemical constituents from caulis of E. schmidtii were identified, including 12 coumarins, 14 chlorogenic acids, 1 tropane alkaloid, 1 amide and 14 esterified glycosides. Conclusion:Chemical constituents in caulis of E. schmidtii can be quickly and fully identified by UPLC-Q-Exactive Focus-MS/MS. Among them, 11 compounds are unambiguously identified by comparing with reference standards, 31 compounds are reported for the first time in this herb, 2 compounds are reported for the first time in Erycibe plants. This paper can provide the important basis for study on pharmacodynamic material base and substitute development of E. schmidtii caulis.

Influence of elicitors on effective constituents and expression of key metabolic enzyme genes in hairy roots of Atropa belladonna / 中草药

Objective To improve the yield of the effective medicinal ingredients of Atropa belladonna, to provide economic and efficient method for the actual production of A. belladonna, therefore to provide a basic reference for the related research on the mechanism of secondary metabolism of medicinal plants. Methods In this study, the influences of four kinds of elicitors, including methyl jasmonate (MJ), AgNO3, salicylic acid (SA), yeast extract (YE), on the accumulation of active components and the expression level of key metabolic enzyme genes, including putrescine N-methyl transferase (pmt), tropinone reductase-I (trI), and hyoscyamine-6-β-hydroxylase (h6h), were studied in hairy roots of A. belladonna. 0.5 g fresh hairy roots of A. belladonna were cultivated in B5 liquid medium. 12 d later, these mediums were replaced with the new medium containing one kind of four elicitors. Hairy roots were taken samples after 2 d to mensurate the fresh weight, dry weight, the content of tropane alkaloids, some physiological indexes, and three key genes expression level. Results MJ inhibited the growth and tropane alkaloids accumulation of hairy roots. The gene expression level of pmt and trI also decreased compared with control group (CK). The contents of tropane alkaloids and the expression level of pmt, trI and h6h were all increased compared with CK by the treatment of AgNO3, while the growth of hairy rootswas inhibited; SA contributed to the increased content of hyoscyamine, but with no obvious influence on growth of hairy roots. As to YE, the content of tropane alkaloids and the expression level of pmt, trI, h6h were all increased correspondingly. Further more, YE was benefit for the growth of hairy roots. Conclusion Elicitors had selective influence on growth and tropane alkaloids accumulation in hairy roots of A. belladonna. The best elicitor on accumulation of tropane alkaloids was AgNO3. YE could effectively improve of the growth of hairy and contents of tropane alkaloids. This study concluded that these elicitors influence the secondary metabolism of A. belladonna by regulating and controlling the expression level of some genes of key metabolic Enzyme, which could provide an effective method to enhance the medicinal composition in the culture of hairy roots of A. belladonna.

Mechanism exploration on nitrogen metabolism and secondary metabolism in Atropa belladonna hairy roots treated with yeast extract / 中国中药杂志

In order to study the mechanism of nitrogen metabolism and secondary metabolism in Atropa belladonna hairy roots treated with yeast extract, yeast extract（YE） was added to the culture medium. Then the changes of physiological and biochemical indexes of A. belladonna hairy roots after treatment with YE were detected. The results are as follows，the activity of key enzymes of nitrogen metabolism changed differently. Compared with the control group (CK), the activity of nitrate reductase (NR) and glutamine synthetase (GS) were significantly increased, while the activity of glutamate dehydrogenase (GDH) was not changed significantly. The content of nitrate nitrogen, ammonium nitrogen had a significant decrease,but the content of soluble protein, free amino acid, total nitrogen are significantly more than CK. Moreover, YE treatment led to the increase of the content precursor amino acids (ornithine and arginine) and precursor putrescine in secondary metabolic pathways of A. belladonna. The expression level of gene putrescine N-methyl transferase (pmt), tropinone reductase-I (trI) and hyoscyamine 6-β-hydroxylase(h6h) all increased in a different rate caused by YE treatment, which eventually led to the increase of the yield of tropane alkaloids. The yield of hyoscyamine and scopolamine were 3.09 and 1.85 folds than that of CK after 16 days treatment time. The results indicated that YE can induce more synthesis of tropane alkaloids by increasing the activity of key enzymes in nitrogen metabolism to provide more synthetic materials for secondary metabolism, meanwhile it regulated the expression level of some genes of key metabolic enzyme to accelerate secondary metabolism.

Cloning and characterization of an aromatic amino acid aminotransferase (ArAT) gene involved in tropane alkaloid biosynthesis from Hyoscyamus niger / 药学学报

Tropane alkaloids are anticholinergic drugs widely used clinically. Biosynthesis of tropane alkaloids in planta involves a step of transamination of phenylalanine. Based on the sequenced transcriptomes of lateral roots and leaves of Hyoscyamus niger, we found three annotated aromatic amino acid aminotransferases, which were respectively named HnArAT1, HnArAT2 and HnArAT3. Sequence analysis showed that HnArAT3 had highest similarity with the reported Atropa belladonna AbArAT4, which was involved in tropane alkaloid (TA) to provide the precursor of the phenyllactic acid moiety. Tissue expression pattern analysis indicated that HnArAT3 was specifically expressed in lateral roots, where is the organ synthesizing tropane alkaloids. Then, method of virus induced gene silencing (VIGS) was used to characterize the function of HnArAT3 in H. niger. Gene expression analysis given by real-time quantitative PCR showed that all the transgenic lines had lower expression levels of HnArAT3 than the non-transgenic control, and HPLC analysis of alkaloids demonstrated significant decrease in the contents of hyoscyamine, anisodamine and scopolamine in planta. These results suggested that HnArAT3 was involved in the phenyllactic acid branch of TA biosynthetic pathway. Molecular cloning and functional identification of HnArAT3 laid the foundation for further understanding of TA biosynthesis and metabolic regulation, and also provided a new candidate gene for engineering biosynthetic pathway of tropane alkaloids.

Cloning and expression analysis on arginine decarboxylase genes in Atropa belladonna / 中草药

Objective: To clone the full-length cDNAs encoding arginine decarboxylase (ADC) from Atropa belladonna, and to characterize the genes at the bioinformatics and expression levels. Methods: cDNAs were used as templates, the full-length cDNAs of ADC in A. belladonna was cloned through rapid amplification of cDNA ends (RACE) technique; Bioinformatics analysis of AbADC genes was performed by BLAST and PBIL on line. The expression levels of the two AbADC genes in different tissues as well as under two different types of stresses were detected based on qPCR analysis. Results: Two ADC genes, namely AbADC1 and AbADC2, were cloned from A. belladonna. AbADC1 was 2 817 bp in length that encoded 712 amino acids with the highest identity of 89% with ADC from Solanum tuberosum; The full-length cDNA of AbADC2 was 2 992 bp in length that encoded 715 amino acids with the highest identity of 90% with ADC from Datura stramonium. The expression level of AbADC1 was significantly higher in secondary roots than that in any other organ, while the AbADC2 expression level was higher in main roots than that in other detected organs. The transcriptional levels of both AbADC1 and AbADC2 were not affected under salinity stress; AbADC2 expression decreased under cold stress, while AbADC1 did not. Conclusion: The cloning and characterization of the cDNAs encoding ADC from A. belladonna are reported for the first time, which provides the new candidate genes for engineering biosynthetic pathway of tropane alkaloids.

Advances in Biosynthesis and Metabolic Engineering of Tropane Alkaloids / 世界科学技术-中医药现代化

Tropane alkaloids,such as hyoscyamine (or its more stable racemate atropine) and scopolamine,are remembered as the oldest drugs in medicine with wide pharmaceutical applications clinically for their mydriatic,antispasmodic,anticholinergic,analgesic and sedative properties.Presently,the supply of tropane alkaloids are entirely rested on their isolation from plant materials,yet the low contents of tropane alkaloids in plants raise the requirement to breed new varieties of high-yield tropane alkaloids and improving the production of tropane alkaloids in hairy root cultures or plants by biotechnology has be a research focus in the field of secondary metabolism.For the past decade,there have been important progresses on biosynthesis of tropane alkaloids and their molecular biology,some new pathway genes have been identified and many achievements have also been acquired in metabolic engineering of tropane alkaloids.This review summarized the recent advances in above aspects,and the problems and orientations of future research are also discussed and proposed.

Changes of alkaloids in Tibetan medicine Przewalskia tangutica during tissue culture and differentiation of its stem rapid propagation system / 中草药

Objective: To establish the stem rapid propagation culture system of Prezewalskia tangutica using aseptic seedling of P. tangutica as materials. Methods: The stems from one month aseptic seedling were inoculated on Muragshige and Skoog media supplemented with different phytohormones to select the optimum medium for inducing differentiation of the adventitious buds and roots. Furthermore, the contents of four active alkaloids were measured with HPLC. Results: The optimum medium was MS+6-BA 2.0 mg/L+NAA 0.5 mg/L for inducing the propagation of the adventitious buds. The adventitious roots could be induced on MS+NAA 0.5 mg/L media. The inductivity was 71.67%. The HPLC data suggested that the content of total alkaloids is highest in root (141.25 μg/g). The content of total alkaloids in callus was lower than those in roots, stems, and leaves. Conclusion: The contents of total alkaloids in different fractions of P. tangutica seedlings are different, and the proportion of different alkaloids in total alkaloids is different, which indicates that the formation and dedifferentiation of organ can influence the contents of alkaloids in P. tangutica seedlings.