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.

Effect of the rolB gene on phenotypic development and tropane alkaloids biosynthesis in Atropa belladonna / 药学学报

The rol genes on pRiA4 plasmid of Agrobacterium rhizogenes are potent genes that promote secondary metabolism. Molecular breeding of Atropa belladonna can be conducted by introducing rol genes to increase tropane alkaloids (TAs) content in A. belladonna. In this study, the rolB gene was overexpressed in A. belladonna plants to study the effect of rolB gene on the biosynthesis of TAs. The phenotype, TAs content and expression levels of key enzyme genes in the pathway of TAs biosynthesis of transgenic A. belladonna were analyzed. The results showed that transgenic A. belladonna had developed root system, enlarged leaves, increased leaf fresh weight, deepened leaf color, enlarged flowers, changed flower shape, reduced pistil height and decreased pollen vitality. The content of TAs in the stems of transgenic A. belladonna was significantly higher than that of the control, and the contents of scopolamine, anisodamine, hyoscyamine can reach 2.11-2.91, 1.23-2.37 and 4.88-5.20 times of the control, respectively. Compared with the control group, the expressions of key enzymes putrescine N-methyltransferase (PMT), type III polyketide synthase (PYKS), tropinone reductase I (TRI), aromatic amino acid aminotransferase 4 (ArAT4), UDP-glycosyltransferase 1 (UGT1) and hyoscyamine 6-β-hydroxylase (H6H) in the TAs biosynthesis pathway were up-regulated, and the expression of tropinone reductase II (TRII) as a metabolic shunting gene was down-regulated. The results indicated that rolB gene enhanced TAs synthesis ability in roots and accumulation in stems of A. belladonna by enhancing metabolic flow of TAs synthesis pathway and weakening the metabolic shunt of competing pathway. This study laid a foundation for molecular breeding of A. belladonna with high-yield TAs content using rolB gene.

Selective extraction of dimethoate from cucumber samples by use of molecularly imprinted microspheres / 药物分析学报

Molecularly imprinted polymers for dimethoate recognition were synthesized by the precipitation polymerization technique using methyl methacrylate (MMA) as the functional monomer and ethylene glycol dimethacrylate (EGDMA) as the cross-linker. The morphology, adsorption and recognition properties were investigated by scanning electron microscopy (SEM), static adsorption test, and competitive adsorption test. To obtain the best selectivity and binding performance, the synthesis and adsorption conditions of MIPs were optimized through single factor experiments. Under the optimized conditions, the resultant polymers exhibited uniform size, satisfactory binding capacity and significant selectivity. Furthermore, the imprinted polymers were successfully applied as a specific solid-phase extractants combined with high performance liquid chromatography (HPLC) for determination of dimethoate residues in the cucumber samples. The average recoveries of three spiked samples ranged from 78.5% to 87.9% with the relative standard deviations (RSDs) less than 4.4% and the limit of detection (LOD) obtained for dimethoate as low as 2.3μg/mL.

Progress in the studies on neuronal nitric oxide synthase inhibitors / 药学学报

Nitric oxide (NO), which is involved in the regulation of the cardiovascular system, nervous system, immune system, reproductive system, digestive system and other physiological activities, is an important biological substance with activity. Under normal physiological conditions, neuronal nitric oxide synthase (nNOS) can precisely regulate the nervous system NO production, release, diffusion and inactivation processes. But an excess of NO associates with the development of cerebral ischemia, Alzheimer's and Parkinson's psychosis nervous system diseases, while inhibition of nNOS activity can regulate the content of NO in vivo, and produce a therapeutic effect on some of the nervous system diseases. This review mainly describes the structure and regulation of nNOS and recent developments of small molecule inhibitors of nNOS.

Chest CT features and outcome of necrotizing pneumonia caused by Mycoplasma pneumoniae in children (report of 30 cases) / 中华儿科杂志

<p><b>OBJECTIVE</b>To summarize the chest CT features and outcome of necrotizing pneumonia (NP) caused by Mycoplasma pneumoniae in children and to review the changes of common inflammatory parameters in NP patients to help clinicians understand the proper timing of CT scan.</p><p><b>METHOD</b>The imaging data from 30 cases of Mycoplasma pneumoniae pneumonia in NP group and 24 cases with non-necrotizing Mycoplasma penumoniae pneumonia (control group) were analyzed retrospectively. The changes of common inflammatory parameters in NP group and control group were compared.</p><p><b>RESULT</b>(1) The chest CT findings of NP (30 cases): 28 cases showed unilateral pneumonia, and 20 cases showed single lobar consolidation, 10 cases had multiple lobes involvement; pulmonary cavities were seen in 27 cases. There were decreased enhancement areas in the consolidation (22 cases). (2) The dynamic changes of CT signs during follow-up: The CT scan performed during the 1 - 2 months after onset of disease (23 cases) showed that pulmonary consolidation in 2 cases (9%) were absorbed, 18 cases (78%)had cavities in lung, 16 cases (70%) had pleural thickening, 2 cases (9%) atelectasis and 1 case (4%) bronchopleural fistula;the CT scan performed during the 2 - 3 months after onset of disease (11 cases) showed pulmonary consolidation in 7 cases (64%) were absorbed, 10 cases (91%) pleural thickness, 7 cases (64%) with cavities in lung, 5 cases (45%) atelectasis, 2 cases (18%) pulmonary lobe cysts and 1 case bronchopleural fistula. The CT scan performed at 3.5 years of disease course (10 cases) showed that there were no pulmonary consolidation in any of the cases, 4 cases had atelectasis, 4 cases had pulmonary cysts, and 1 case had band-like scars. (3) There were significant differences between NP group and control group in the maximum peripheral blood WBC, proportion of neutrophil and C-reactive protein(CRP, mg/L) (P < 0.01, 0.01, 0.001, respectively), and there was significant difference between the 2 groups in the duration of fever, abnormal WBC(d) and CRP(d) (P < 0.001).</p><p><b>CONCLUSION</b>The chest CT features of NP caused by Mycoplasma pneumoniae in children were single lobular consolidation in most cases, NP had decreased parenchymal enhancement and cavity in the consolidation, and recovery was slow, the outcome included recovery, atelectasis or lobar cystic degeneration. The clinicians should pay more attention to the common inflammatory parameters when they suspect the Mycoplasma pneumoniae pneumonia is progressing into necrosis and make correct decision for chest CT examination.</p>

Determination of fluoroquinolones, sulfonamides,and tetracyclines multiresidues simultaneously in porcine tissue by MSPD and HPLC-DAD / 药物分析学报

An efficient method is provided to detect simultaneously some important veterinary drugs from different classes in highly complex animal tissue matrix.This method using matrix solid-phase dispersion (MSPD) and high performance liquid chromatography (HPLC) with diode array detection (DAD) is developed to effectively determine two fluoroquinolones (enoxacin and lomefloxacin),two sulfonamides (sulfanilamide and sulfamethoxazole) and one tetracycline (tetracycline) simultaneously in porcine tissues.In the process,MSPD methodology was used to treat samples,washed by n-hexane to remove lipid,eluted the analytes with acetonitrile-dichloromethane (1∶1,v/v).Solvent acetonitrile and solvent acetic acid (0.1％) were combined in a gradient.HPLC-DAD analysis of the tissue samples was performed within 15min at a flow rate of 1.0mL/min.The results showed that a recovery at 0.1,0.5 and 1.0 μg/g fortification levels ranged from 80.6％ to 99.2％ with satisfactory relative standard deviations (RSDs) (below 6.1％.n=3) and the limits of quantitation (LOQ) ranged from 7 μg/kg to 34 μg/kg in porcine tissues.Utilization of the method in successfully simultaneous analysis of porcine tissue incurred with veterinary drug multiresidues is described.

A rapid method for the determination of dopamine in porcine muscle by pre-column derivatization and HPLC with fluorescence detection / 药物分析学报

A rapid method has been developed based on the sample preparation procedure named as QuEChERS (Quick,Easy,Cheap,Effective,Rugged and Safe),combined with reversed-phase high performance liquid chromatography with fluorescence detector and C18 column after precolumn derivatization using o-phthalaldehyde and 2-mercaptoethanol to determine dopamine in porcine muscle.Methanol and deionized water (0.1％ acetic acid,v/v) with a ratio of 60∶40 was used as mobile phase.The flow rate was 0.8 mL/min and dopamine was eluted within 15 min.The linearity range was 0.003-8 μg/mL with r=0.9992.The detection limit for dopamine was 4 μg/kg and the quantification limit was 9 μg/kg.Recovery studies were carried out at 0.1,0.5 and 1.0 mg/kg fortification levels and the average recoveries obtained ranged from 90.4％ to 98.2％ with relative standard deviations between 3.5％ and 8.1％.The method was found to be suitable for detection of dopamine in animal product tissues at the maximum residue level.

A rapid method for the determination of dopamine in porcine muscle by pre-column derivatization and HPLC with fluorescence detection / 药物分析学报

A rapid method has been developed based on the sample preparation procedure named as QuEChERS （Quick, Easy, Cheap, Effective, Rugged and Safe）, combined with reversed-phase high performance liquid chromatography with fluorescence detector and C18 column after precolumn derivatization using o-phthalaldehyde and 2-mercaptoethanol to determine dopamine in porcine muscle. Methanol and deionized water （0.1% acetic acid, v/v） with a ratio of 60：40 was used as mobile phase. The flow rate was 0.8 mL/min and dopamine was eluted within 15 min. The linearity range was 0.003-8 μg/mL with r=0.9992. The detection limit for dopamine was 4 μg/kg and the quantification limit was 9 μg/kg. Recovery studies were carried out at 0.1, 0.5 and 1.0 mg/kg fortification levels and the average recoveries obtained ranged from 90.4% to 98.2% with relative standard deviations between 3.5% and 8.1%. The method was found to be suitable for detection of dopamine in animal product tissues at the maximum residue level.

Studies on chemical constituents from the root of Polygonatum kingianum / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the anti-HIV constituents from the root of Polygonatum kingianum.</p><p><b>METHOD</b>The compounds were isolated by column chromatography on silica gel, Sephadex LH-20, MCI-gel CHP-20P and their structures were determined on the basis of their spectroscopic evidence including IR, MS and NMR data.</p><p><b>RESULT</b>13 compounds were isolated, of which nine compounds were identified as liquiritigenin, isoliquiritigenin, 4', 7-dihydroxy-3'-methoxyisoflavone, (6aR, 11aR)-10-hydroxy-3, 9-dimethoxypterocarpan, 5-hydroxymethyl-2-furancarboxaldehyde, salicylic acid, n-butyl-beta-D-fructopyranoside, n-butyl-beta-D-fructofuranoside, n-butyl-alpha-D-fructofuranoside.</p><p><b>CONCLUSION</b>Compounds 1-6 were obtained from this plant for the first time.</p>

DETERMINING THE CONTENT OF JASMINODIN IN MEI RONGCAPSULE WITH THIN LAYER CHROMATOGRAPHY / 药物分析学报

Objective Jasminodin in Mei Rong capsule was determined by the method of thin layer chromatography. Methods The sample jasminodin was extracted with methanol and then the extract was chromatographed on silica gel GF254 plate with chloroform-methanol-water (10:3:1) as mobile phase and the chromatograms were scanned by using Shimadzu CS-930 dual wavelength TLCS in zegzag scanning mode at λs 370nm and λR 250nm.Results Calibration graph was rectilinear between 2.7μg～16.2μg per spot for the jasminodin. Conclusion Satisfatory results were obtained with the average recovery ratio of 99.76% ,RSD 2.27%.

EFFECT OF METHIONINE ENKEPHALIN ON MIGRATION OF MACROPHAGES FROM MICE WITH IMPAIRED LIVER / 药物分析学报

Objective To observe the effects of methionine enkephalin (M-Enk) on migration of macrophages from mice with impaired liver and its immunomodulatory mechanisms. MethodsLiver of mice was impaired by feeding CCl4 and macrophage migration inhibitory factor (MMIF) was produced by Con A-stimulated spleen lympho- cytes. Inhibition of macrophage migration was measured in reaction system by adding M-Enk. Results Migration of macrophages in both liver-impaired and control group were suppressed by MMIF, but the suppression might be re- versed by adding 1 μmol/L M-Enk (P＜0. 05). M-Enk could significantly inhibit in vitro both of the combination of MMIF with macrophages and production of MMIF from lymphocytes (P＜0. 01). Macrophages from liver-imparied group showed a higher sensitivity compared to the control group (P＜0. 05). ConclusionThe study suggests that opi- oid peptieds play an important role in the modulation of the immune response under stress as liver impairment.