Supervisor(s):Chinese Pharmaceutical Association Sponsor(s):Tianjin Institute of Pharmaceutical Research；Chinese Pharmaceutical Association ISSN:0253-2670 CN:12-1108/R
Chinese Traditional and Herbal Drugs is supervised by Chinese Pharmaceutical Association and sponsored by Tianjin Institute of Pharmaceutical Research and Chinese Pharmaceutical Association. Launched in 1970, the journal is an academic journal with a broad scope in publishing research papers, brief reports, reviews, dissertations, and special treatises on the recent achievements of basic study, production, quality control, and clinic application on traditional Chinese medicine and Chinese materia medica.
The journal is included in CA, JST and CSCD.
Objective To screen and identify the dominant strains which produce fibrinolytic enzyme during the processing of Sojae Semen Praeparatum (SSP, Dandouchi in Chinese). Methods SSP was prepared according to the Chinese Pharmacopoeia (2020 edition), and samples were taken at different time points during the fermenting process of SSP. The casein plate method and fibrin plate method were used to screen the fibrinolytic enzyme-producing microorganisms in samples at different time points. The fibrinolytic enzyme-producing microorganisms were inoculated in the designated liquid medium to obtain single strain fermentation broth, and fibrin plate method was used to measure the fibrinolytic activity of the fermentation broth. The DNA sequences of fibrinolytic enzyme-producing bacteria and fungi were amplified using 16S rDNA and 18S rDNA universal primer by PCR respectively. The amplified products were sequenced, and the sequencing results were identified through NCBI homology comparison. Molecular biological identification was done by phylogenetic tree constructed by MEGA 4.1. Results Three types of fibrinolytic enzyme-producing bacteria were screened out and identified in this study. They were Bacillus subtilis, Stenotrophomonas maltophilia and Micrococcus, respectively. The result of fibrin plate method showed that the fermentation broth of S. maltophilia had the highest fibrinolytic activity, reaching 527.49 IU/mL. Conclusion There are fibrinolytic enzyme-producing dominant microorganisms existing in the fermenting process of SSP and the thrombolytic effect of SSP is worthy of further study. This study lays the foundation for revealing the formation mechanism of fibrinolytic enzyme in the fermentation process of SSP.
Objective The discrete element method(DEM) was used to simulate the angle of repose of Chinese medicine extract powders. The contact parameters between particles and between particles and geometry were calibrated. Methods The licorice extract powder, the extract powders of Angelicae Pubescentis Radix(Duhuo), microcrystalline cellulose(MCC) and ethyl cellulose(EC) were taken as the research objects. DEM was performed based on the Hertz-Mindlin with JKR Cohesion contact model and particle scaling. The Plackett-Burman design was used to screen out the critical contact parameters that had a significant impact on the simulation measurement of the angle of repose. Then, the steepest climbing design was used to determine the best area of critical contact parameters. After that, a regression model between the contact parameters and the simulated angle of repose was established according to the Box-Behnken test results, and the best contact parameter values were optimized and verified. Results The critical contact parameters selected were particle-particle rolling friction coefficient, particle-particle restitution coefficient and particle-stainless steel restitution coefficient. The calibration range of the built regression model was from 33.30° to 43.64°. The absolute values of the relative error between the simulated values and the experimental values of the angle of repose for four powders were all less than 2.0%, indicating that the established calibration method was accurate and reliable. Conclusion This article proved the feasibility of calibrating the DEM micromechanical parameters of the Chinese medicine particle system through the macroscopic physical parameter, and laid the foundation for accurate simulation of Chinese medicine pharmaceutical processes like powder mixing and conveying.
Objective To clone the tryptophan synthase gene named as BcTSB (GenBank accession number AYM45644.1) involved in the synthesis pathway of indole alkaloids from Baphicacanthus cusia, meanwhile, the bioinformatics analysis and expression analysis were also performed. Method The open reading frame(ORF) of BcTSB gene was obtained by the database of prophase Baphicacanthus cusia transcriptome. The function of the BcTSB gene was preliminarily predicted by a series of bioinformatics tools. The entire protein-coding cDNA of BcTSB was cloned into the prokaryotic expression vector pET32a, then the recombinant plasmid was transformed into E. coli BL21(DE3) cells, with IPTG induction. SDS-PAGE was used to investigate the situation of expression. The expression of the gene in root, stem and leaf was determined by using real-time PCR(qRT-PCR). Results The open reading frame(ORF) of cloned BcTSB gene was 1 452 bp, and encoding 483 amino acids, it was predicted by bioinformatics analysis as hydrophilic protein, being located in the chloroplasts. Bioinformatics analysis of the amino acid sequence showed that the molecular weight of encoded protein was 52 kDa, because prokaryotic expression vector pET32a contained 18 kDa label, SDS-PAGE results showed that a protein band at 70 000 was in consistent with molecular weight of the predicted protein. The QRT-PCR revealed that BcTSB gene was expressed in different tissues of B. cusia, the expression level of BcTSB in stems was much higher than that in roots and leaves. Conclusion In this study, BcTSB gene of B. cusia was cloned and its expression was analyzed successfully, which laid an experimental foundation for further study on the function and regulation of the gene.
Objective In order to provide a scientific basis for the quality control of Kunxian Capsules (KC), HPLC characteristics chromatogram combined with multi-components determination were established. Methods The analysis was performed on Agilent Zorbax SB-C18 column (250 mm × 4.6 mm, 5 μm), using acetonitrile and 0.1% phosphoric acid solution as the mobile phase at a flow rate of 0.8 mL/min for gradient elution, the column temperature was 33 ℃, and the detection wavelength was 270 nm. The fingerprints of 15 batches of KC were established and evaluated by the similarity evaluation system of TCM (2012A version), hierarchical cluster analysis and discriminant analysis of partial least squares. Furthermore, the content of hyperoside, epimedin A, epimedin B, epimedin C, icariin and baohuoside Ⅰ were determined. Results The HPLC fingerprint with 21 common peaks of KC was established, and the similarities of samples were over 0.9. The linearity relationships separated with hyperoside, epimedin A, epimedin B, epimedin C, icariin and baohuosideⅠwere good, and the contents of the above-mentioned components in 15 batches of preparations were 2. 817–7.527, 7.287–9.103, 8.730–18.675, 33.377–70.371, 35.297–50.291 and 4.059–9.079 mg/g, respectively. Conclusion The combination methods of HPLC characteristic chromatograms and simultaneous determinations of multiple components are rapid, simple and reproducible, which can provide methodological reference for the quality control of KC.
Objective: Sialic acid (SA)-modified chlorogenic acid (CA) liposomes (CA-SAL) were prepared by the response surface design to investigate the in vitro cytotoxicity and uptake. Methods: CA-SAL was prepared by an improved reverse-phase ethanol injection method. Sephadex G-50 columns were used to separate liposomes and the free drug. Mass concentrations of the drug were determined by the HPLC method, and encapsulation efficiency was calculated. With encapsulation efficiency and drug loading as investigation indexes, the Box-Behnken response surface design experiments were conducted to optimize the formulation and preparation process of CA-SAL. The MTT method was used to evaluate the cytotoxicity of CA-SAL to human lung cancer cells A549. An inverted fluorescence microscope was used to observe the CA-SAL uptake by A549 cells. Results: The optimized preparation conditions were listed as follows: the ratio of phospholipid to drug: 15:1; hydration temperature: 60 °C; ultrasonic power: 400 W. The average particle size, polydispersity index (PDI), Zeta potential, encapsulation efficiency and RSD of CA-SAL were calculated to be (90.13 ± 0.51) nm, 0.16 ± 0.01, (−25.3 ± 0.5) mV, 57.8%, and 0.1%, respectively. MTT results showed that CA-SAL was better than CA-CL in inhibiting the proliferation of A549 cells. Cellular uptake experiments showed that the CA-SAL uptake by A549 cells was much higher. Conclusion: CA-SAL was prepared based on response surface optimization and displayed small particle size and good stability. SA-modified liposomes can enhance cellular uptake and in vitro cytotoxicity.
Objective To optimize the infiltration process of Astragalus (Astragalus membranaceus var. mongholicus) medicinal materials by Box-Behnken response surface method. Methods Based on the HPLC-DAD-ELSD and response surface design method, the qualified rate of decoction pieces, the content of index components and bending inspection were used as comprehensive inspection indicators, and the three factors of infiltration were selected for response surface experimental design to optimize the infiltration process of Astragalus medicinal materials parameter. Results The best infiltration process was as following: infiltration temperature was 20 ℃, with water addition of 1:0.988 for 6 h. Under this process, the qualified rate of Astragalus pieces was 95.81%, the content of calycosin-7-glucoside was 0.072%, and the content of astragaloside IV was 0.276 %. Combining fingerprint analysis and heat map analysis, the material basis of A. membranaceus var. mongholicus changed during the infiltration process. The infiltration parameters should be strictly controlled during the infiltration process to ensure uniform quality of the pieces. Conclusion The optimized Astragalus medicinal material infiltration process is stable and feasible with good reproducibility, which can provide a reference for the mass production process development of Astragalus medicinal slices.
Objective To investigate the mechanism of cytoskeletal recombination and migration inhibition induced by wangzaozin A, an ent-kaurane diterpene, in A549 cells. Methods The effects of wangzaozin A on cytotoxicity, cell morphology, cytoskeleton, and protein expression as well as cell migration were detected in A549 cells by using MTT, microscope observation, Western blotting, immunofluorescence assay and scratch assay. Results Wangzaozin A induced significant changes in cell morphology at 24, 48, and 72 h, including increased pseudopods, stretched pseudopods, and flattened nucleus in A549 cells. Moreover, microtubules and keratin fibers networks in A549 cells also showed obvious rearrangement, which indicated the cytoskeleton had gone through a continuous recombination process. Further, wangzaozin A significantly increased the phosphorylation of extracellular regulated protein kinase (ERK), microtubule-associated protein 4 (MAP4), keratin 8 (K8) (P < 0.05, 0.01), while wangzaozin A-induced phosphorylation of MAP4 and K8 were suppressed in A549 cells treated with ERK inhibitors U0126 (P < 0.05, 0.01); wangzaozin A inhibited the migration of A549 cells in a concentration- and time-dependent manner. Conclusion Wangzaozin A can upregulate the phosphorylation of MAP4 and K8 by activating ERK signaling pathway, which can significantly increase the dynamics of MTs and KFs, disturb the dynamic balance of the cytoskeleton, and inhibit the migration of A549 cells.
