OBJECTIVE To construct molecularidentification of Fritillaria taipaiensis and its relatives based on Indel marker. METHODS One hundred and three chloroplast genomes of 13 Fritillaria species were downloaded from GenBank and were subsequently used to screen Indel marker based on sequence alignment. Then, in accordance with the conserved upstream and downstream of Indel marker, a pair of specific primers was designed accordingly, by which a method including routine PCR and electrophoresis was constructed. Furthermore, the specificity, sensitivity, and optimal reaction temperature of the method were assessed. RESULTS One deletion of 137 bp was found in the accD-psaI spacer region of F. taipaiensis chloroplast genome according to the multiple sequence alignments, which was defined as the Indel marker for F. taipaiensis. The constructed method exhibited high specificity due to the fact that only F. taipaiensis exhibited a band of 302 bp after routine PCR and electrophoresis. The limit of detection for F. taipaiensis DNA template was 0.239 ng·μL^-1, and the optimal melting temperature (T_m) value was 58 ℃. CONCLUSION This accurate, quick and cost-affordable method provides not only identification of F. taipaiensis and its related species, but also technical reference for identification of other traditional medicines.

OBJECTIVE To study and establish sequence characterized amplifiedregion(SCAR) molecular marker for identifying the original plants and related plants of Rabdosia Herba, and provide a new molecular identification method for the original plants of Rabdosia Herba. METHODS A total of 29 populations of the original plants and related plants of Rabdosia Herba were collected, and inter-simple sequence repeat (ISSR) molecular marker was used to perform PCR amplification on the genomic DNA of the representative population of Rabdosia Herba to obtain ISSR-specific segments. After the ISSR-specific fragments were recovered, purified, cloned and sequenced, SCAR primers were designed based on the sequencing results, which were directly used for the efficient identification of the original plants of Rabdosia Herba. RESULTS Fourteen pairs of ISSR primers were used to perform PCR amplification on 18 representative samples of original plants and related plants of Rabdosia Herba, and 4 specific segments were obtained and successfully transformed into ISSR-SCAR molecular markers. These molecular markers can be used to specifically identify the Rabdosia lophanthoides (Buch. -Ham. ex D. Don) H. Hara, Rabdosia stracheyi (Benth. Ex Hook. f.) Hara, Rabdosia serra (Maxim.) H. Hara, and Rabdosia nervosa (Hemsl.) C. Y. Wu et H. W. Li, respectively. CONCLUSION The established ISSR-SCAR molecular markers produce clear and bright bands with stable results, which can accurately and rapidly identify the original plants and related plants of Rabdosia Herba, providing a new method for the identification of the original plants of Rabdosia Herba.

OBJECTIVE To study the in vitro release behavior and anti-tumor effect of paclitaxel-loaded rhein conjugate (PTX/TPGS-CR) micelles. METHODS The in vitro drug release behavior of PTX/TPGS-CR micelles in different release media was investigated by dialysis method with Taxol^® as the control. The cytotoxicity of PTX/TPGS-CR micelles on mouse breast cancer 4T1 cells was evaluated by MTT assay with Taxol^® as a control. The effect of PTX/TPGS-CR micelles on the cell cycle and apoptosis of human cervical carcinoma HeLa cells was detected by flow cytometry. The effect of PTX/TPGS-CR micelles on the migration of HeLa and 4T1 cells was investigated by scratch experiments. RESULTS The results of in vitro release assay showed that 50.84% and 71.38% of PTX in PTX/TPGS-CR micelles were released in pH 7.4 phosphate buffer (PBS) within 12 and 72 h, respectively, 79.19% of PTX in PTX/TPGS-CR micelles were released in pH 5.8 PBS which simulated the tumor microenvironment within 12 h, and almost all of PTX in the micelles was released within 72 h. The results of the cytotoxicity assay showed that PTX/TPGS-CR micelles had a strong inhibitory effect on the proliferation of 4T1 cells and displayed a time-and-concentration-dependent characteristic, with IC₅₀ of 0.321 9 nmol·mL^-1 at 72 h. PTX/TPGS-CR micelles could induce early apoptosis and late apoptosis in HeLa and 4T1 cells. PTX/TPGS-CR micelles can induce HeLa cells arrest in G2/M phase (arrested by PTX) and G0/G1 phase, which inhibited cell division and proliferation. PTX/TPGS-CR micelles can inhibit the migration of HeLa and 4T1 cells and reduce tumor metastasis. CONCLUSION PTX/TPGS-CR micelles have good pH-responsive drug release characteristics, which can release drugs slowly in the blood environment and rapidly in the tumor microenvironment. PTX/TPGS-CR micelles could inhibit tumor cells proliferation and migration and promote cell apoptosis. PTX/TPGS-CR micelles have a good tumor treatment potential.

OBJECTIVE To investigate the nebulization characteristics of Houning inhalation solution (HNIS)in vitro and to provide the reference for the clinical administration. METHODS The delivery rate (DR),delivered amount (DA) and aerodynamic particle size distribution (APSD) in vitro of HNIS were determined by means of respiratory simulator and the next generation impactor(NGI)with the syringin as the indication component and compared among the different types of nebulizers, compressors, atomization modes and breathing patterns. RESULTS In the same atomization condition and under the adult breathing pattern,the DR were between 23.3-52.2 μg·min^-1, the DA were between 178.8-353.2 μg and the deposition fraction in simulated throat (DFST) was about 2.1% for the jet nebulizers, the DR were between 3.2-21.2 μg·min^-1, the DA were between 12.9-82.7 μg and the DFST were about 0.7% for the ultrasonic nebulizers, the DR were between 7.2-16.5 μg·min^-1, the DA were between 93.5-217.2 μg and the DFST were about 0.1% for the mesh nebulizers, the mass median aerodynamic diameter (MMAD ) were between 2.2-3.6 μm. In the same atomization condition and under the child breathing simulation,the DR were between 1.5-24.3 μg·min^-1, the DA were between 26.3-215.4 μg. CONCLUSION The nebulization characteristics of HNIS are significant affected by the patterns and brands of the atomiztion equipment, suggesting that the atomization system should be selected according to the requirement in the clinical. Under the child breathing pattern, with the DA and DR reduced, the dosage and atomization time should be adjusted according to the age and pulmonary function of the patients to ensure the safety and efficiency of clinical medication.

OBJECTIVE To study the pharmacokinetics of polymyxin B (PB) in sepsis patients and to provide evidence for its rational clinical use. METHODS Sepsis patients were given intravenous PB, every 12 h (q12 h), 4 consecutive doses. Blood samples were collected before each dose and 0.5, 1, 2, 3, 6, 9, 12 h after the fifth dose. The plasma concentrations of PB were determined by UHPLC-MS/MS method. The main pharmacokinetic parameters were calculated by non-compartment model, and a limited sampling model was established with sparse blood concentration data points. RESULTS A total of 18 patients were included. The main pharmacokinetic parameters of PB were as follows: area under the concentration-time curve for the 24 h exposure after the fifth dose(AUC_{0-24 h}) was (63.33±30.88) mg·h·L^-1, plasma concentrations prior to the fifth dose(ρ₀) was (2.01±1.54) mg·L^-1, post the fifth dose peak plasma concentration(ρ_max) was (6.90±4.22) mg·L^-1, plasma concentrations of 12 h after the fifth dose(ρ₁₂) was (1.64±1.04) mg·L^-1, average steady-state plasma concentration(ρ_ss,av) was (2.64±1.29) mg·L^-1, trough concentrations after the first, second and third doses were (3.35±2.50) (2.74±1.60) and (1.76±1.10) mg·L^-1, respectively. The recommended two-point regression equation is AUC_{0-12 h}=2.08+1.22ρ₁+9.41ρ₆. CONCLUSIONS There are individual variabilities in the pharmacokinetic parameters of PB in patients with sepsis. The loading dose of the first dose can reach the steady-state concentration as soon as possible, and the PB concentration point 1,6 h after administration can be used to estimate AUC_{0-24 h}. It is suggested to monitor PB-AUC_{0-24 h} to guide the adjustment of PB dosage in adult patients with sepsis.

OBJECTIVE To establish the HPLC fingerprint of Forsythiae Fructus and develop a multi-component quantitative analysis method based on quantitative analysis of multi-components by single-marker(QAMS). METHODS The fingerprints of 47 Forsythiae Fructus samples from four provinces, including Henan, Shandong, Shanxi and Shaanxi, were established by high performance liquid chromatography (HPLC), while their similarity was evaluated. Chemical pattern recognition of Forsythiae Fructus was studied by hierarchical cluster analysis (HCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA). The content of forsythoside B, forsythoside A, hyperin, arctiin, phillyrin and phillygenin were determined by using QAMS, and the differences of samples from different producing areas were analyzed. RESULTS The fingerprints of 47 Forsythiae Fructus samples were established with similarities ranged from 0.785 to 0.989, and a total of 30 common peaks were identified. According to HCA, the samples were divided into three groups. OPLS-DA analysis can effectively distinguish Forsythiae Fructus from several producing areas (Q²>0.5). There was no significant difference in the content of 6 components in 47 batches of samples by QAMS and external standard method (ESM) (P>0.05), but there was significant difference in the content of active components in samples from different origin (P<0.01). The average contents of forsythoside B, forsythoside A, hyperin, arctiin, phillyrin and phillygenin in samples from different provinces were, Henan(4.50、20.58、0.52、2.39、7.46、1.76 mg·g^-1), Shandong(0.90, 9.26、0.36、1.39、3.84、3.87 mg·g^-1), Shanxi(0.82、8.74、0.29、1.16、2.64、1.75 mg·g^-1), Shaanxi(5.18、55.10、0.88、3.36、13.11、9.25 mg·g^-1). CONCLUSION The chemical composition of Forsythiae Fructus samples from different locations was similar, but the origin factors had a significant effect on the content of active ingredients, and the samples from Shaanxi province had the highest content. The HPLC fingerprint combined with the simultaneous determination of multi-component content analysis method established in this study is stable and reliable. The fingerprint combined with the chemical pattern recognition can provide a basis for the identification of origins and quality evaluation of Forsythiae Fructus.

OBJECTIVE To establish a method for simultaneous determination of 8 aminoglycoside antibiotics (AGs) including kanamycin, gentamycin C1, gentamycin C2, gentamycin C1a, tobramycin, neomycin, isepamicin and etimicin by LC-MS/MS with instant library searching. METHODS Eight AGs were separated by Shiseido PC HILIC (2.0 mm×100 mm, 3 μm) column, with 0.5% formic acid aqueous solution-0.1% formic acid acetonitrile solution as mobile phase and 0.4 mL·min^-1 of flow rate,and procedure of the gradient elution was performed. The screening method of 8 AGs was established by the scanning mode of multiple reaction monitoring (MRM). Typical mass spectrograms of 8 AGs were collected by collision energy expansion (CES) function and enhanced ion (EPI) scanning mode, and added them to the already established mass spectralibrary of antibiotics searching. RESULTS The established method has the advantages of short analysis time, strong specificity and high sensitivity. The screening and qualitative analysis of 8 AGs can be realized within 10 min. Three batches of the INSA insulin product were detected by this method, and kanamycin that was less than the detection limit of 0.05 ng·mg^-1 were not detected in above samples. CONCLUSION In this study, the detection method of 8 AGs established by LC-MS/MS with instant library searching can quickly screen and accurately determine trace AGs.

OBJECTIVE To establish a UPLC-MS/MS method for simultaneous determination of 14 components in Shenmai granules, and analyze the quality differences of red ginseng feed from different manufacturers. METHODS Shimadzu Shim-pack gist C₁₈(2.1 mm×100 mm,2 μm) chromatographic column with acetonitrile-water as the mobile phase were used to determine the contents of 14 saponin components in 71 batches of Shenmai Granules. And then analyzed the main material basis affecting the difference in red ginseng feeding in Shenmai granules combined with chemometrics and evaluated the quality of red ginseng feeding in Shenmai granules from different manufacturers combined with EW-TOPSIS. RESULTS The concentrations of 14 components had a good linear relationship (r≥0.998 5); and the average recovery rate was 95.6%-105.0%(RSD<4.7%). According to the analysis of chemometrics and EW-TOPSIS, the samples produced by the three manufacturers were grouped into three categories, among which the quality of red ginseng feeding by manufacturer B was generally good, and the red ginseng feeding by manufacturer C was adulterated. CONCLUSION This method is specific, sensitive, efficient, and reliable, and can be used for the quality control of Shenmai granules.

OBJECTIVE To explore the key technical considerations in the drugs design of clinical trials for delaying the progression of chronic kidney disease. METHODS By reviewing domestic and foreign literature, and considering the characteristics of the disease, drug development and review experience, the important design considerations for clinical trials evaluating drugs that delay the progression of chronic kidney disease were suggested in this article. RESULTS and CONCLUSION We recommend an overall trial design that incorporates randomization, double-blinding, and placebo/standard treatment as control measures. The study population should be representative of a wide range of individuals who reflect the intended objectives of the trial. Inclusion criteria and efficacy evaluation should take into account etiology, staging, risk factors for disease progression, and combined treatments. We suggest the composite endpoints based on clinical events as primary efficacy endpoints,while the average change rate in glomerular filtration rate is recommended for early or slowly progressing chronic kidney disease cases. Safety evaluation indicators should not only adhere to general standards but also consider potential safety issues related to investigational drugs as well as safety signals associated with natural disease progression by setting corresponding safety assessment parameters for underlying diseases.

OBJECTIVE To discuss the characteristics of research and development on the new drugs of renal disease and technical considerations envolved. METHODS By investigating the advance of the regulatory agencies home and abroad and the related literature,the nephrotic drugs of the last decade in the market and in research were summerized domestically and overseas,the characteristics of research and key technical points were analyzed and relevant suggestions were put forward. RESULTS and CONCLUSIONS Ten new drugs have been approved by China,European Union and United States since 2013. The applications of the new drugs on this field has continued to increase in the recent 5 years, mainly on the chronic kidney disease and IgA nephropathy. To better promote the drug innovation development in this field, the following aspects should be paid attention to: The first is to further strengthen the basic scientific research about the pathogenesis of diseases, and screen the potential new drugs with new target and mechanism, so that make it possible to delay and even reverse the progression of the disease. The second is to better evaluate the patients beneifts by developing scientific and rational clinical outcome assessment tools with the basis of the patients' needs. The third is to choose the representative population by the combination of epidemiology, natural history, the feature of drugs and the aim of clinical trials since the pathology of renal diseases is complicated. The fourth, early management of renal disease is crucial. So it's of great significance to explore, develop and validate the surrogate endpoints since the disease progresses slowly and the investigation often requires both long study cycle of several years or even longer and a larger sample size. The surrogate indicators should be selected prudently. The sponsors are suggested to communicate with the regulatory agency when necessary. The fifth, a sufficent safety study can be very supportive to the general evaluation of benefit-risk balance.