Object To study the callus culture of Gynura segetum (Lour.) Merr., so as to lay the foundation for the rapid propagation of its breeds.Methods The explants used in culture were root, stem and leaf of G. segerum. The MS basal medium containing different plant hormones was applied. Results The best explants were leaf of G. segetum. The medium induced callus was MS basal medium containing 1 0 mg/L NAA and 4 0 mg/L 6 BA and the callus induction rate was 64% . The sprouting rate of the callus transplanted to fitting differential medium was 86 7%.Conclusion The rapid propagation of G. segetum can be carried out by inducing callus of the plant.

Objective To investigate the impact of multi-drug transporters including P-glycoprotein (PGP) and multi-drug resistance associated protein (MRP) on concentration of lamotrigine in the extracellular fluid in hippocampus of epilepsy rat models induced by pilocarpine, and to deduce the multi-drug resistance mechanisms in refractory epilepsy. Methods The epilepsy rat models were established by repeated administration (by ip) of pilocarpine. A microdialysis probe was placed into the hippocampus of the epileptic rats and dialysate was collected at five time-points from 30--150 minutes after systemic injections of lamotrigine (10 mg/kg). The concentration of lamotrigine in the extracellular fluid in the hippocampus was determined by high-performance liquid chromatography (HPLC). Then PGP inhibitor verapamil and MRP inhibitor probenecid was added individually through microdialysis probe and the concentration of lamotrigine was detected again. Results Compared with control group (0. 41 ± 0. 10 in 60 minutes, 0. 50 ±0.04 in 90 minutes, 0. 39 ±0. 09 in 120 minutes and 0. 30±0.06 in 150 minutes), verapamil significantly increased the concentration of lamotrigine in extracellular fluid of the hippocampus 60--150 minutes (0. 65 ±0. 11, 0. 84 ± 0. 09, 0. 70± 0. 09 and 0. 58 ± 0. 08 respectively) after injection (F value were 5.01, 8.61, 10. 23 and 7.89, all P < 0. 05) and probenecid also enhanced the concentration of lamotrigine 90--150 minutes (0. 75 ± 0. 09, 0. 58±0. 10 and 0. 49±0. 07) after injection (F = 6. 58, 4. 56, 4. 75, all P < 0. 05). Conclusions Penetration of lamotrigine through blood-brain barrier in pilocarpine induced epilepsy rats is restricted by PGP and MRP, resulting in decreased concentration of lamotrigine in the extracellular fluid of the hippocampus. Therefore, increasing expression of PGP and MRP in brains of epilepsy patients might be an important mechanism involved in multi-drug resistance in refractory epilepsy.

Objective To study the effect of inhibitors of the multidrug transporters including P-glycoprotein (PGP)and multi-drug resistance-associated protein(MRP)on the regulation of concentration of oxcarbazepine in the extra-cellular fluid of the hippocampus after pilocarpine induced seizures in rats.To investigate whether oxcarbazepine are sub-strate for PGP and MRP and whether brain expressions of PGP and MRP are involved in muhidrug resistance mechanisms of refractory epilepsy.Methods The epileptic rats model were established by repeated peritoneal injection treatment with pi-locarpine.Thirty-two Wistar rats were divided into four groups:control group,pilocarpine epileptic model group,verapamil treated group and probenecid treated group.At 30,60,90,120 and 150 min following systemic injection of oxcarbazepine (80 mg/kg),dialysate was collected and the concentration of oxcarbazepine in the extracellular fluid of hippocampus was determined by microdialysis and high-performance liquid chromatography technique.Results After systemic injection of oxcarbazepine,the concentration of oxcarbazepine in extracellular fluid of the hippocampus during 90～150 min(1.26±0.09、0.93±0.10)were much higher in verapamil treated group than in pilocarpine epileptic model group(0.87±0.06、0.66±O.04)(P<0.05)and the concentration of oxcarbazepine in the hippocampus during 60～150 min(1.07 4±0.11、1.32±O.13、1.02±0.10、0.87±0.08)were higher in probenecid treated group than in pilocarpine epileptic model group (0.81±0.08、0.87±0.06、0.66.4±0.04、0.58±0.06)(P<0.05).Conclusions Oxcarbazepine are substrates for PGP and MRP and,penetration of oxcarbazepine through blood-brain barrier are restricted by PGP and MRP.Increased expres-sions of PGP and MRP in brain maybe involved in the mechanisms of multidrug resistance of refractory epilepsy.

Objective To explore the protective effects of a new military functional food NB-5 on psychological stress-induced oxidative stress .Methods Rat whiskers were completely removed to induce the oxidative stress , and the concen-trations of MDA and protein carbonyl in various organs were detected to study the damage to membrane lipid and protein . Rats were fed with NB-5 for 4 weeks, and the oxidative stress was induced by whisker cutting .Biochemical marks men-tioned above were detected to explore the protective effects of NB-5.Results and Conclusion Lipid and protein peroxida-tion occurred in the brain , heart, liver, spleen and kidney after whisker removal due to emotional stress , while the catalase ( CAT) activity decreased significantly in these organs except the spleen .In this experiment model , NB-5 showed a good free radical scavenging activity to reduce the lipid and protein peroxidation among whisker -cutting rats fed with NB-5 in ad-vance.So NB-5 can serve as a good food for soldiers in case of emergency incidents .

Plants provide an immense reservoir of natural secondary metabolites. Secondary metabolites and those involved enzymes accumulate in various compartments in specific plant tissues. The biosynthesis of diverse groups of secondary metabolites is often complicated, tightly controlled via network interconnections, metabolite levels, metabolite channeling and multi-enzyme complexes, and so on. Secondary metabolite profiles could be genetically altered by two strategies, i.e. single gene modification and multiple gene modification; which thus has opened a feasible and prospective platform for secondary chemicals production in plant.
Gene Expression Regulation, Plant ; Phytochemicals ; biosynthesis ; genetics ; Plants ; genetics ; metabolism ; Plants, Genetically Modified ; genetics ; metabolism ; Secondary Metabolism ; genetics ; Transformation, Genetic

OBJECTIVE To establish the fingerprints of Ligusticum sinense from different habitats ，screen differential components and determine their contents. METHODS Using Z-ligustilide as reference ，HPLC fingerprints of 12 batches of L. sinense were established by using Similarity Evaluation System of Chromatographic Fingerprints of TCM （2012 edition）；common peaks were identified and their similarities were evaluated. Cluster analysis （CA），principal component analysis （PCA）and orthogonal partial least squares-discriminant analysis （OPLS-DA）were performed to screen differential components with variable importance in the projection （VIP）＞1 as standard ；meanwhile，the contents of above differential components were determined by the same HPLC method. RESULTS There were 17 common peaks in the fingerprints of 12 batches of L. sinense ，and their similarities ranged 0.989-1.000. A total of 9 common peaks were identified ，i.e. chlorogenic acid （peak 1），ferulic acid （peak 2）， senkyunolide Ⅰ（peak 7），coniferyl ferulate （peak 9），E-ligustilide（peak 13），senkyunolide A （peak 14），Z-ligustilide（peak 17）. CA results showed that 12 batches of L. sinense were divided into 3 categories，S1-S5（Wuning）were clustered into one category，S6-S8（Ruichang）were clustered into one category ，S9-S12（De’an）were clustered into one category ；the VIP values of peaks 2，13，14 and 17（corresponding to ferulic acid ，E-ligustilide，senkyunolide A ，and Z-ligustilide respectively ）were all greater than 1，respectively. In S 1-S5，S6-S8 and S 9-S12 samples，the contents of ferulic acid were 0.488-0.533，0.603-0.658 and 0.415-0.433 mg/g，respectively；senkyunolide A were 1.184-1.295，1.450-1.588 and 1.307-1.377 mg/g，respectively；E-ligustilide were 0.118-0.125，0.130-0.135 and 0.223-0.229 mg/g，respectively；Z-ligustilide were 7.200-7.681，8.076-8.643 and 4.508-4.996 mg/g， respectively；the differences between two groups were statisti-cally significant （P＜0.05）. CONCLUSIONS Established ARS-11）；fingerprint is simple and accurate ，and can be used for overall quality evaluation of L. sinense from different habitats by combining with multivariate statistical analysis. Ferulic acid ， senkyunolide A ，Z-ligustilide and E-ligustilide may be the differential components that affect the quality of L. sinense from different habitats ，the contents of the first 3 components in L. sinense from Ruichang are the highest ，and the content of E-ligustilide in samples from De’an is the highest.

ObjectiveTo analyze and predict the potential quality markers （Q-Marker） in the Genuine medicinal materials Jiangxi Aurantii Fructus based on fingerprints and network pharmacology. MethodUltra-high performance liquid chromatography （UPLC） and gas chromatography-mass spectrometry （GC-MS） fingerprints were established for 18 batches of Jiangxi Aurantii Fructus ，combined with chemometric methods to screen out candidate Q-Marker components.Use network pharmacology to construct a "core component-target-pathway" network to predict the Q-Marker and core targets of Jiangxi Aurantii Fructus，and then verify the biological activity of Jiangxi Aurantii Fructus Q-Marker by molecular docking method. ResultThe 18 batches of Jiangxi Aurantii Fructus use UPLC，GC-MS fingerprints combined with chemometric analysis，a total of 9 Q-Marker candidate components were screened out.Through network pharmacological analysis，it is predicted that nobiletin，neohesperidin，meranzin，naringin and D-limonene are the Q-Marker of Jiangxi Aurantii Fructus，acting on the core targets transforming protein p21/H-Ras-1（HRAS），cellular tumor antigen p53 （TP53），mitogen-activated protein kinase 8 （MAPK8），transcription factor AP-1（JUN），glycogen synthase kinase-3 beta（GSK3B），tumor necrosis factor（TNF），cyclin-dependent kinase inhibitor 1（CDKN1A），cAMP-dependent protein kinase catalytic subunit alpha（PRKACA），cysteine aspartate-specific protease-9（Caspase-9），cyclic AMP-responsive element-binding protein 1（CREB1），exerting gastrointestinal motility and antidepressant，anti-inflammatory，anti-tumor，etc.； molecular docking shows that nobiletin，neohesperidin，meranzin，naringin and D-limonene and the selected 10 core targets have good binding ability，reflecting the better biological activity of the Q-Marker of Jiangxi Aurantii Fructus. ConclusionThe Q-Marker of Jiangxi Aurantii Fructus can be comprehensively predicted from the two aspects of volatile and non-volatile components，providing a reference for the quality control of Jiangxi Aurantii Fructus and the further study of its pharmacodynamic mechanism.

L-asparaginase (L-ASN) is widely applied in the treatment of malignant tumor and low-acrylamide food production, however, the low expression level hampers its application. Heterologous expression is an effective strategy to increase the expression level of target enzymes, and Bacillus is generally used as the host for efficient production of enzymes. In this study, the expression level of L-asparaginase in Bacillus was enhanced through optimization of expression element and host. Firstly, five signal peptides (SP_SacC, SP_AmyL, SP_AprE, SP_YwbN and SP_WapA) were screened, among which SP_SacC showed the best performance, reaching an activity of 157.61 U/mL. Subsequently, four strong promoters (P43, P_ykzA-P43, P_Ubay and P_bacA) from Bacillus were screened, and tandem promoter P_ykzA-P43 showed the highest yield of L-asparaginase, which was 52.94% higher than that of control strain. Finally, three Bacillus expression hosts (B. licheniformis Δ0F3 and BL10, B. subtilis WB800) were investigated, and the maximum L-asparaginase activity, 438.3 U/mL, was reached by B. licheniformis BL10, which was an 81.83% increase compared with that of the control. This is also the highest level of L-asparaginase in shake flask reported to date. Taken together, this study constructed a B. licheniformis strain BL10/P_ykzA-P43-SP_SacC-ansZ capable of efficiently producing L-asparaginase, which laid the foundation for industrial production of L-asparaginase.
Bacillus licheniformis/metabolism* ; Asparaginase/genetics* ; Bacillus/genetics* ; Protein Sorting Signals ; Promoter Regions, Genetic/genetics* ; Bacillus subtilis/genetics* ; Bacterial Proteins

Bacitracin is a broad-spectrum cyclic peptide antibiotic, and mainly produced by Bacillus. Energy metabolism plays as a critical role in high-level production of target metabolites. In this study, Bacillus licheniformis DW2, an industrial strain for bacitracin production, was served as the original strain. First, our results confirmed that elimination of cytochrome bd oxidase branch via deleting gene cydB benefited bacitracin synthesis. Bacitracin titer and ATP content were increased by 10.97% and 22.96%, compared with those of original strain, respectively. Then, strengthening cytochrome aa3 oxidase branch via overexpressing gene qoxA was conducive to bacitracin production. Bacitracin titer and ATP content were increased by 18.97% and 34.00%, respectively. In addition, strengthening ADP synthesis supply is also proven as an effective strategy to promote intracellular ATP accumulation, overexpression of adenosine kinase DcK and adenylate kinase AdK could all improve bacitracin titers, among which, dck overexpression strain showed the better performance, and bacitracin titer was increased by 16.78%. Based on the above individual methods, a method of combining the deletion of gene cydB and overexpression of genes qoxA, dck were used to enhance ATP content of cells to 39.54 nmol/L, increased by 49.32% compared to original strain, and bacitracin titer produced by the final strain DW2-CQD (DW2ΔcydB::qoxA::dck) was 954.25 U/mL, increased by 21.66%. The bacitracin titer produced per cell was 2.11 U/CFU, increased by 11.05%. Collectively, this study demonstrates that improving ATP content was an efficient strategy to improve bacitracin production, and a promising strain B. licheniformis DW2-CQD was attained for industrial production of bacitracin.
Bacillus licheniformis ; metabolism ; Bacitracin ; biosynthesis ; Energy Metabolism ; genetics ; Industrial Microbiology ; methods