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??OBJECTIVE To study the plasma protein binding rate of decapeptide in several species. METHODS Ultrafiltration was employed to separate the free and bound decapeptide, acetonitrile was used to precipitate protein, then the plasma concentration of decapeptide by HPLC-MS/MS. RESULTS The plasma protein binding rates of decapeptide at low, middle and high concentrations (50.0, 100.0, and 800.0 ng??mL^-1) were (95.9??1.1)%, (97.40??0.7)% and (94.9??0.6)% in SD rats,(96.8??0.8 )%, (97.8??0.2 )% and (96.9??0.5 )% in Beagle dogs, and (97.3??1.0 )%, (98.6??0.2 )% and (96.2??0.9)% in humans, respectively. The average plasma protein binding rate was 96.2% after subcutaneous administration at 200 ??g??kg^-1 in SD rats. And the average plasma protein binding rate was 97.1% after intramuscular injection at 320 ??g??kg^-1 in Beagle dogs. CONCLUSION Decapeptide has potent binding ability to plasma protein in several species. The plasma protein binding rate of decapeptide is independent of its plasma concentrations.     

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??Dengue fever is one of the most important vector-borne human diseases caused by mosquito vector Aedes aegypti and Aedes albopictus. Dengue virus can cause dengue fever, dengue hemorrhagic fever and dengue shock syndrome. There are no approved drugs for the treatment of dengue disease so far. According to the mechanism of anti-dengue virus(anti-DENV) action, drugs under development for dengue disease can be divided into two categories:viral replication inhibitors and anti-cell factor pathway inhibitors. The former is further divided into DENV entry inhibitors, capsid protein inhibitors, NS3 protein inhibitors, NS5 protein inhibitors, and NS4B protein inhibitors; the latter is further divided into cell receptor inhibitors, lipid synthesis and metabolism inhibitors, and glucosidase inhibitors. The R&D of anti-DENV drugs is facing enormous challenges. Development of effective drugs which can be used for the treatment of four serotypes of dengue has a broad application prospect, and it will bring new hopes for dengue fever prevention and therapy.     

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??OBJECTIVE To synthesize low molecular weight chitosan-acetylcysteine (LMWC-NAC) conjugate and investigate its renal targeting profile and the rapeutic effects in model mice with acute kidney injury (AKI).METHODS NAC was conjugated to LMWC by EDC/NHS reaction and the LMWC-NAC conjugate was identified by ¹H-NMR. The cellular uptake of LMWC-NAC conjugate and megalin receptor involved in this process was investigated in vitro. In addition, the tissue distribution of ICG-labelled LMWC-NAC conjugate was investigated in nude mice. AKI were induced by LPS intraperitoneal injection (20 mg??kg^-1).The parameters including Scr, BUN, inflammatory factors (TNF-?? and IL-1??),and oxidative stress (MDA) were determined and renal histology was observed.RESULTS LMWC-NAC conjugate was successfully synthesized by the amide interaction.The in vitro studies demonstrated that the uptake of LMWC-NAC conjugate was mediated by the megalin receptor on HK-2 cells, and the tissue distribution experiment indicated that LMWC-NAC conjugate was mainly accumulated in the kidney.LMWC-NAC conjugate significantly suppressed Scr, BUN, inflammatory factors and oxidative stress (P<0.01) and improved kidney injury. CONCLUSION LMWC-NAC conjugate showed good renal targeting profile and effect in recovering renal functions, which indicates the potential of LMWC-NAC conjugate as a safe and efficient drug delivery system for the treatment of AKI.     

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??OBJECTIVE To establish dose-related lung inflammatory injury in rats model with intratracheal atomization of lipopolysaccharide (lipopolysaccharides, LPS). METHODS Four groups of 4 rats were subjected to solvent or a single dose of LPS by intratracheal route using a IA-1B-2 inches-microsprayer. The male rats received 200 ??L solvent (control), LPS solutions (15, 5, 0.5 mg??kg^-1). All rats were sacrificed 24 h after dose administration, biochemical analysis and cell counts on bronchoalveolar lavage fluid (BALF) were performed on each rat. Lung, trachea and kidney were examined histologically. Serum chemistry profiles of creatinine, ALB, Na, K, Cl^- were detected. RESULTS Cell counts in BALF showed LPS groups had different degrees of inflammatory reaction. The alkaline phosphatase and total protein concentration were higher in LPS high dose group compared with other groups. In addition, the concentration of TNF-?? increased consistently with LPS dose and has statistical significance compared with the control group. Histopathology findings demonstrated that LPS produced an accumulation of foamy macrophages in the lungs and high degree of inflammation. CONCLUSION The results recommends intratracheally atomizing doses of LPS in rats model produced ranks of lung inflammatory injury.     

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??OBJECTIVE To select the fungi which can induce Dalbergia odorifera to produce active substances. METHODS Twenty-five fungi were inoculated in the stems of six or seven-year-old D. odorifera under natural conditions. Fungi with biological activity to induce D. odorifera producing active substances were obtained through field screening and chemical quality evaluation. The ethanol-soluble extractives and total flavonoids of D. odorifera were analyzed by spectrophotometry and hot maceration method, respectively. RESULTS Six active fungi inducing the formation of heartwood were obtained. CONCLUSION This study is very important for utilizing fungi in the induction of D. odorifera and sustainable utilization of this special medicinal material.     

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目的以壳聚糖为酶触型结肠靶向给药系统的材料,研制可用于结肠靶向的多单元口服给药系统(迷你片),以期为结肠部位疾病治疗的药物输送提供重要参考。方法以结肠癌的治疗药物-吲哚美辛为模型药物,首先制备固体分散体,再采用直接压片法和包衣技术制备尤特奇-壳聚糖双层包衣结肠靶向迷你片,考察靶向迷你片在不同释放介质中的释药行为,采用小动物活体荧光成像技术考察制剂在体内的转运和吸收情况,并以比格犬为动物模型进行药动学研究和生物利用度评价。结果制备的壳聚糖多单元结肠靶向迷你片可以完整形态通过大鼠胃和小肠,并靶向在结肠部位缓慢释放,比格犬体内药动学数据表明,自制结肠靶向迷你片的释药时间显著延长,血药浓度平稳。结论本实验制备尤特奇-壳聚糖双层包衣多单元迷你片给药系统具有较好的结肠靶向性和缓释效果,可为治疗结肠疾病的制剂开发提供重要参考。     

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??OBJECTIVE To observe the effect of chlorogenic acid on the secretion of inflammatory cytokines and regulation mechanism of P38MAPK, NF-??B signaling pathway in human hepatic stellate cells induced by TGF-??1. METHODS Different concentrations of CGA worked in normal and activated hepatic stellate cells to make sure the appropriate drug concentration.The exponential growth phase cells were randomly divided into normal HSC group, normal HSC+CGA group, after cultured 48 h, the cells were cultured with 0??50??100 mg??L^-1 CGA for 24 h; HSC(TGF-??1) group?? HSC(TGF-??1 + CGA) group: after 24 h, the cells were induced by 10 ??g??L^-1 TGF-??1 for 24 h, and then cultured with 0, 50, 100 mg??L^-1 CGA for 24 h. The expression of ??-SMA protein was detected by immunocytochemistry, the expression of p-P38, P65 protein was detected by Western-blot, the expression of TNF-??, IL-6 mRNA was detected by real time quantitative PCR, and the content of TNF-??, IL-6 in the supernatant was detected by ELISA method. RESULTS The appropriate concentrations of CGA were 50 and 100 mg??L^-1, these concentration has no effect on normal HSC(P>0.05); after stimulation by TGF-??1, the expression of ??-SMA, p-P38, P65, TNF-??, IL-6 was increased(P<0.01), when activated HSC cells were treated with 50 and 100 mg??L^-1 CGA, the expression of ??-SMA, p-P38, P65, TNF-??, IL-6 was decreased(P<0.05, P<0.01). CONCLUSION CGA can inhibit the proliferation of activated HSC, regulate the secretion of inflammatory factors such as TNF-??, IL-6 by P38MAPK and NF-??B signaling pathway, inhibit the occurrence of liver fibrosis.     

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??As a model organism, Drosophila melanogaster has the characteristics of short life cycle, high fecundity and high gene conservation. At present, Drosophila melanogaster has been widely used in the studies of pharmacology and pathogenesis, including aging, neurodegenerative disease, insomnia, tumor and other fields, which show potential values in accelerating the speed of drug screening and target discovery. In this paper, the advantages of Drosophila as a model organism, the applications and limitations of Drosophila in pharmacological studies were reviewed.     

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??OBJECTIVE To establish an HPLC-UV-ESI-MSⁿ method for the study of impurity profile of amoxicillin and clavulanate potassium tablets. METHODS Agilent 1100 LC/MSD Trap liquid chromatography-mass spectrometry was used, and the column was Shim pack CLC-ODS RP18(4.6 mm??250 mm, 5 ??m). The mobile phase A was 20 mmol??L^-1 ammonium acetate (pH adjusted to 6.0), and the mobile phase B was 20 mmol??L^-1 ammonium acetate-acetonitrile (20??80) (pH adjusted to 6.0). Gradient elution was performed at a flow rate of 1.0 mL??min^-1. ESI source was used. Positive and negative ion scan was conducted with a scanning range of m/z 100-1 500. The nebulizing pressure was 275.8 kPa, dry gas flow was 9 L??min^-1, and post-column diversion ratio was 1??5. Some related substances were identified by comparing the retention time in the chromatography, [M+H]⁺ spectrum and MS² spectrum with those of the reference substances, while the others which do not have reference substances were deduced or speculated by analyzing the MS² or MSⁿ fragmentation with the help of a rule summarized from the MS² fragmentation of amoxicillin, clavulanic acid and system suitability impurity reference substances. RESULTS A total of 15 related substances were separated and characterized including nine known impurities like amoxicilloic acid, amoxicillin dimer, etc. and six unknown impurities. CONCLUSION The method can be applied in the identification and qualitative analysis of the related substances in amoxicillin and clavulanate potassium tablets and is helpful for the quality control and optimization of the synthetic process.     

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??OBJECTIVE To establish a high performance size exclusion chromatography(HPSEC) method for the separation and analysis of polymers in cefotaxime sodium and cefotaxime sodium for injection, and determine the structures of the impurities by LC-MS. METHODS HPSEC was performed by using Sepax SRT SEC-150(7.8 mm??300 mm, 5 ??m)column. The mobile phase was 0.1 mol??L^-1 disodium hydrogen phosphate and 0.1 mol??L^-1 phosphate buffer solution. The flow rate was 0.8 mL??min^-1, the detection wavelength was set at 235 nm, the injection volume was 10 ??L, and the column temperature was maintained at 35 ??. The concentration of polymers was quantified by external standard method. The LC-MS/MSⁿ system conditions were as following: the mobile phase was 20 mmol??L^-1 amonium acetate, the flow rate was 0.8 mL??min^-1, ESI source with positive and negative ion scan was utilized, the scanning range was m/z 200-1 600, and the post-column diversion ratio was 1??4. RESULTS Eight impurity peaks were obtained in total; the resolutions were all greater than 1.5. The linear range of cefotaxime was 1-100 ??g??mL^-1(r=1.000 0). The RSD repeatability was 1.2%(n=6). The limit of detection was 0.2 ??g and the limit of quantitation was 0.4 ??g. Three polymers were identified by LC-MS. CONCLUSION The HPSEC method can be used for the quantitative and qualitative analyses of individual polymer impurities. It is also sensitive for the control of polymers in cefotaxime.     

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??OBJECTIVE To establish a gradient elution method for determination of related substances of azithromycin for injection. METHODS Gradient elution was used for the analysis. A C₁₈ (CAPCELL PAK MG??, 4.6 mm??250 mm,5 ??m)column was used. The mobile phase A consisted of 0.05 mol??L^-1 K₂HPO₄ solution (pH 8.2, adjusted with 20% phosphoric acid)-acetonitrile (45??55), and the mobile phase B was methanol. The flow rate was 1.2 mL??min^-1, and the detection wavelength was set at 210 nm. The column temperature was maintained at 30 ??. The gradient elution program was as follows: 0-35 min, A:75%??95%; 35-64 min, A:75%; 64-65 min, A:95%??75%; 65-71 min, A:75%. RESULTS Azithromycin, near peaks and known impurities were well separated from each other. All acid radicals of azithromycin for injection did not influence the determination of related substances of azithromycin. CONCLUSION This method is more specific than the exising method for determination of related substances of azithromycin, which can more effectively control the quality of the drug.     

注射用磺苄西林钠的杂质谱研究

目的 建立高效液相色谱串联高分辨质谱检测器的方法,研究注射用磺苄西林钠的杂质谱并进行来源归属。方法 采用Agilent 1290 HPLC-6538 Q-TOF高效液相色谱质谱联用仪,流动相为10 mmol·L^-1甲酸铵溶液-8 mmol·L^-1甲酸铵溶液(体积分数80%乙腈溶液作溶剂)=87∶13;色谱柱为Ultimate XB C₁₈(4.6 mm×250 mm,5 μm);流速1.0 mL·min^-1;质谱检测器采用电喷雾离子源(ESI),负离子扫描模式;数据采集范围m/z 50~1 000,离子源前进样分流比2∶1,毛细管电压3.5 kV,锥孔电压65 V,喷雾气压310 kPa,干燥气(氮气)流量12 L·min^-1,干燥气温度325 ℃,碎裂电压150 V。通过分析主成分与未知杂质多级质谱行为的相关性以及主成分的合成工艺推定其结构。结果 将5个厂家样品中的主要杂质归纳为14个未知杂质,解析其质谱碎片进行结构推测和来源归属。结论 研究结果对注射用磺苄西林钠的质量控制和工艺评价具有指导意义。     

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??OBJECTIVE To compare the HPLC-PAD method and HPLC-ELSD method for determination of related substances in spectinomycin hydrochloride for injection, and determine 31 batches of samples by the two methods. METHODS The following conditions were used for both methods:the analytical column was Apollo C₁₈ column (4.6 mm??250 mm,5 ??m), mobile phase was 0.1 mol??L^-1 trifluoroacetic acid solution, the column temperature was maintained at 30 ??, and the sample volume was 20 ??L. For the PAD detection, gold electrode (3 mm)was used as the detection electrode, Ag-AgCl as reference electrode, titanium alloy as counter electrode, and integrated amperometric detector was employed with four waveform detection potential. For the ELSD detection, the drift tube temperature was set at 110 ??, and the carrier gas flow rate was 2.6 L??min^-1, with gain of 1. RESULTS The two methods were consistent in the species and number of detected impurities. The detection limits of the PAD method and ELSD method were 2.4 and 72.8 ng, respectively (S/N=3). For the PAD method there existed a direct linear relationship in the concentration range of 0.15-150 ??g??mL^-1, while a double logarithmic linear relationship was shown in the concentration range of 17-170 ??g??mL^-1 for the ELSD method. When 31 batches of samples were determined for related substances by the two kinds of methods, the contents of impurity D and E were basically the same, but there were significant differences in the contents of impurity A and (4R)-dihydrospectinomycin. After correction, the contents of impurity A and total impurities determined by the PAD method were consistent with those by the ELSD method. CONCLUSION The two methods can both effectively detect the related substances of spectinomycin to achieve the purpose of controlling related substances. When using HPLC-PAD method, the impurity A and (4R)-dihydrospectinomycin should be adjusted by using response factor or the reference substances. The ELSD method needs to use double log linear regression.     

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??OBJECTIVE To rapidly identify the related substances in doxycycline hyclate tablets and investigate the possible degradation pathways of doxycycline hyclate solution by 2D-LC-IT-TOF/MS. METHODS Firstly,the chromatography was carried out using the 1stD InertSustain^TM C₁₈(4.6 mm??150 mm,5 ??m) column and a mobile phase containing a mixture of buffer solution(0.25 mol??L^-1 ammonium acetate solution-0.1 mol??L^-1 ethyldiaminetetraacetic acid disodium: triethyiamine=100??10??1)-acetonitrile(85??15). Solution pH was adjusted to 8.8 with glacial acetic acid. Then a InertSustain^TM C₁₈(2.1 mm??50 mm,2 ??m) column was used with 0.1% formic acid-water as mobile A and 0.1% formic acid-acetonitrile as mobile B in a gradient elution mode in 2ndD. Electrospray ionization (ESI) source was tested in both positive and negative ion modes. Nebulized gas flow was 1.5 L?? min^-1. Dry gas flow was 10 L?? min^-1. The desolvation tube temperature was kept at 200 ??. Related substances were characterized according to multi-level MS behaviors. The 2D-LC-IT-TOF/MS method was employed to identify the structures of impurities in forced degradation solutions and illuminate the degradation pathways of doxycycline hyclate solution. RESULTS A total of eight related substances were detected in doxycycline hyclate tablets. Four of them had a content of more than 0.1%, and their structures were identified to be 4-epidoxycycline, metacycline, ??-epidoxycycline and 2-acetyl-2-decarbamoyldoxycycline. The solution of doxycycline hyclate easily degraded under alkaline condition and generated an open loop compound taken off the keto group. The solution was sensitive to heat, and generated 4-epidoxycycline with a little amount of 4-epi-6-epidoxycycline; but the solution was stable under illumination and acidic condition. CONCLUSION The established method is suitable for rapid identification of impurities in doxycycline hyclate, which can be applied as a useful analytical tool for quality control and drug process optimization of doxycycline hyclate.     

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??OBJECTIVE To develop a suitable HPLC method combined with component preparation to analyze the related substances and effective contents in bacitracin.METHODS The separation was performed on Agilent HPLC system with a Diamonsil Plus C₁₈ column (4.6 mm??250 mm, 5 ??m) using 50 mmol??L^-1 ammonium formate (pH adjusted to 4.0 with formic acid) as mobile phase A and acetonitrile as mobile phase B at a flow rate of 1 mL??min^-1 under gradient elution. The detection wavelength was set at 254 nm. The HPLC method was carried out by adjusting the pH of ammonium formate solution, the ratio of mobile phase and the solvent. Furthermore, the specificity, linearity, precision, repeatability, stability and durability were studied. The known components like bacitracin A, B1/B2/B3, C1/C2/C3 and F were collected by preparative HPLC according to the HPLC method for bacitracin in USP 40. They were positioned in ammonium formate-acetonitrile HPLC system.RESULTS The established HPLC method was verified to be suitable for analyzing the related substances and effective contents in bacitracin. Baseline separation between the known components and adjacent impurities was achieved, especially the B1/B2. The elution positions of the known component were clearly defined. The contents of the known components in different batches of bacitracin were compared. More detailed sample information was provided in this research.CONCLUSION The HPLC method can simultaneously analyze the related substances and effective contents in bacitracin, and is suitable for the quality control of the product.     

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??OBJECTIVE To establish an HPLC-MS/MS method for determination of ASC-J9 in rat blood. METHODS After liquid-liquid extraction, ASC-J9 was separated on a Symmetry C₁₈colume, with mobile phase of acetonitrile-water containing 0.1% formic acid and 10 mmol??L^-1 ammonium formate.The flow rate was 1.0 mL??min^-1, mass shunt was 0.4 mL??min^-1, and column temperature was main tained at 35 ??. Quantification was performed in positive ion multiple-reaction-monitoring(MRM) mode. RESULTS The calibration curve of ASC-J9 had good linearity in the concentration range of 3.54-1 180 ng??mL^-1. The extraction recovery rate was within 83.19% to 87.27%,and the intra-day and inter-day RSDs were both less than 8.89%. CONCLUSION This method is specific, sensitive and suitable for determination of ASC-J9 in rat blood.
     

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??OBJECTIVE To establish a detection method based on a novel type of two-dimensional liquid chromatographic system (2D-LC-UV) for determination of paraquat in poisoned patients' urine, and assess the methodology and characteristics of the two-dimensional chromatography. METHODS 2D-LC-UV contains 1st and 2nd liquid chromatography and FLC transferor. Urine sample of 100 ??L was directly injected without pretreatments, and paraquat was on-line concentrated and primarily separated on the 1^st Aston SX1 column, then trapped on Aston SX column and separated by Aston SX1 column. The mobile phase of the 1st liquid chromatography, trapping column and 2nd liquid chromatography were acetonitrile-2 mol??L^-1 ammonium acetate binary solution(1:10, V:V, 1.0 mL??min^-1), pure water and 2.5 mol??L^-1 ammonium acetate solution-methanol-acetonitrile ternary system (5:3:1, V:V:V, 1.0 mL??min^-1), respectively. The column temperature was maitained at 40 ??, and the UV adsorption wave length was set at 285 nm. RESULTS The chromatographic system was capable of processing 500 ??L urine. Paraquat was transferred and separated completely in the two-dimensional system with linear calibration curve over the concentration range of 20-20 000 ng??mL^-1 (r=0.999 9). The urine matrixes from different samples had little interference effect. The chromatographic balance time was less than 15 min, and the calibration curve was stable for more than 90 d. CONCLUSION Because of its accuracy, stability, automation, and independence of specialized technical personnel, the established chromatographic method is ideal for quick test of paraquat in poisoned patients, providing scientific basis for clinical treatment of patients with paraquat poisoning.     

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??OBJECTIVE To establish a qualitative and quantitative HPLC method for the determination of impurity ??in tebutaline sulfate.METHODS The Kromasil C₁₈column(4.6 mm??150 mm,5 ??m)was used as the analysis column;buffer solution [dissolving 4.23 g of sodium hexanesulfonate in 770 mL of 0.050 mol??L^-1 ammonium formate solution (pH 3)]-methanol (77:23) was used as the mobile phase. The flow rate was 1.0 mL??min^-1, the column temperature was maitained at 30 ??, the detection wavelength was set at 276 nm,and the injectiong volume was 20 ??L.Area normalization method with correction factor was used for the quantitative analysis of the impurity ??. RESULTS Under the separation condition,the impurity ?? was completely separated from the principal components. The calibration curve showed good linearity in the concentration range of 0.10-579 ??g??mL^-1(r??1.000 0). The correction factor was 3.6.CONCLUSION The area normalization method with correction factor developed in the paper can be used for the qualitative and quantitative analysis of the impurity ??in terbutaline sulfate,which can not only solve the problem of the availability of impurity reference standards,but also reflect the actual contents of impurity.The method provides an efficient and convenient method for quality control of terbutaline sulfate.