EFFECTS OF INHIBITORS ON I-METHYLADENINE INDUCED MATURATION OF STARFISH OOCYTES

抗氧化剂吡咯烷二硫代氨基甲酸有助于提高卡铂对宫颈癌SiHa细胞化疗的敏感性闫亚妮;吴静;张颖;徐丹;潘荣;刘变利【摘要】Objective To study the effect of antioxidant pyrrolidine dithiocarbamate (PDTC) in promoting the chemosensitivity of human cervical cancer cell line SiHa to carboplatin and the possible mechanisms. Methods Human cervical carcinoma SiHa cell lines were cultured in vitro. They were treated with different concentrations of PDTC, carboplatin or PDTC combined with carboplatin for different durations. Then the inhibition of SiHa cells growth was detected by MTT assay; flow cytometry was used to analyze the apoptosis and cell cycle. The cellular distribution and protein level of NF-κBp65 were detected by immunocytochemical staining. Results PDTC could significantly inhibit the proliferation of SiHa cells in time-dependent and dose-dependent manners (P<0.01). Compared with carboplatin alone, PDTC (12.5 μmol/L) combined with carboplatin could significantly inhibit the proliferation of SiHa cells(P<0.01). The apoptosis rate of SiHa cell differed significantly between the drug group and the control group (P<0.01); the difference between PDTC + carboplatin combination group and carboplatin alone group was significant (P<0. 01). PDTC promoted G0/G1 phase of cell cycle arrest while carboplatin promoted G2/M phase of cell cycle arrest. Interestingly, G2/M phase was further arrested when carboplatin was added. The cells of S phase were also decreased. NF-κBp65 was mainly located in the cytoplasmof SiHa cell in the control group. The expression of NF-κBp65 in SiHa cell was transferred from cytoplasm to nucleus after 24-hour treatment with carboplatin, which increased the activity, whereas PDTC could inhibit the activity. Conclusion The activity of NF-κB p65 in SiHa cell was increased when treated with carboplatin. Even at a lower concentration, carboplatin combined with PDTC (12.5 μmol/L) resulted in a more significant growth inhibition and apoptosis of SiHa cell than carboplatin alone. PDTC can increase the chemosensitivity of cervical cancer SiHa cell to carboplatin.%目的探讨抗氧化剂吡咯烷二硫代氨基甲酸(PDTC)提高人宫颈癌SiHa细胞对卡铂化疗的敏感性及其可能的机制.方法体外培养SiHa细胞,分为空白组、PDTC组、卡铂组和PDTC+卡铂联合组.MTT检测细胞生长抑制率、流式细胞术检测细胞凋亡率和细胞周期、免疫细胞化学方法观察了NF-κBp65在细胞质和细胞核中的表达变化.结果 PDTC能够有效地抑制宫颈癌SiHa细胞的增殖,呈时间和剂量依赖关系；小剂量PDTC(12.5μmol/L)和卡铂联合应用与单用卡铂相比,能明显增加细胞生长抑制率(P＜0.01).流式细胞仪检测示,各药物组与对照组比较及PDTC+卡铂联用组与单用卡铂组比较宫颈癌SiHa细胞凋亡率的差异有显著性(P＜0.01).SiHa细胞经PDTC作用后G0/G1期比例较空白组明显增加,卡铂单用组使细胞周期阻滞于G2/M期,PDTC+卡铂组使细胞周期进一步阻滞于G2/M期,同时降低S期比例.免疫细胞化学显示,NF-κBp65在宫颈癌SiHa中主要表达于细胞质中,卡铂诱导24h 后细胞质中的NFκBp65转移至细胞核,其活性增强,PDTC能够抑制此作用.结论卡铂能够诱导NFκBp65的活化,小剂量PDTC(12.5 μmol/L)和卡铂联合应用与单用卡铂相比,能明显增加细胞生长抑制率和细胞凋亡率,提高宫颈癌SiHa细胞对卡铂的敏感性.【期刊名称】《西安交通大学学报（医学版）》【年(卷),期】2011(032)005【总页数】5页(P610-614)【关键词】富颈肿瘤;卡铂;吡咯烷二硫代氨基甲酸;核转录因子κB;凋亡【作者】闫亚妮;吴静;张颖;徐丹;潘荣;刘变利【作者单位】西安交通大学医学院第二附属医院妇产科,陕西西安,710004;西安交通大学医学院第二附属医院妇产科,陕西西安,710004;西安交通大学医学院第二附属医院妇产科,陕西西安,710004;西安交通大学医学院第二附属医院妇产科,陕西西安,710004;西安交通大学医学院第二附属医院妇产科,陕西西安,710004;西安交通大学医学院第二附属医院妇产科,陕西西安,710004【正文语种】中文【中图分类】R737.33宫颈癌是全球妇女恶性肿瘤中仅次于乳腺癌的第2个最常见的恶性肿瘤。

HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationFLUOXETINE HClC17H18F3NO•HClM.W. = 345.79CAS — 59333-67-4STABILITY INDICATINGA S S A Y V A L I D A T I O NMethod is suitable for:ýIn-process controlþProduct ReleaseþStability indicating analysis (Suitability - US/EU Product) CAUTIONFLUOXETINE HYDROCHLORIDE IS A HAZARDOUS CHEMICAL AND SHOULD BE HANDLED ONLY UNDER CONDITIONS SUITABLE FOR HAZARDOUS WORK.IT IS HIGHLY PRESSURE SENSITIVE AND ADEQUATE PRECAUTIONS SHOULD BE TAKEN TO AVOID ANY MECHANICAL FORCE (SUCH AS GRINDING, CRUSHING, ETC.) ON THE POWDER.ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationTABLE OF CONTENTS INTRODUCTION........................................................................................................................ PRECISION............................................................................................................................... System Repeatability ................................................................................................................ Method Repeatability................................................................................................................. Intermediate Precision .............................................................................................................. LINEARITY................................................................................................................................ RANGE...................................................................................................................................... ACCURACY............................................................................................................................... Accuracy of Standard Injections................................................................................................ Accuracy of the Drug Product.................................................................................................... VALIDATION OF FLUOXETINE HCl AT LOW CONCENTRATION........................................... Linearity at Low Concentrations................................................................................................. Accuracy of Fluoxetine HCl at Low Concentration..................................................................... System Repeatability................................................................................................................. Quantitation Limit....................................................................................................................... Detection Limit........................................................................................................................... VALIDATION FOR META-FLUOXETINE HCl (POSSIBLE IMPURITIES).................................. Meta-Fluoxetine HCl linearity at 0.05% - 1.0%........................................................................... Detection Limit for Fluoxetine HCl.............................................................................................. Quantitation Limit for Meta Fluoxetine HCl................................................................................ Accuracy for Meta-Fluoxetine HCl ............................................................................................ Method Repeatability for Meta-Fluoxetine HCl........................................................................... Intermediate Precision for Meta-Fluoxetine HCl......................................................................... SPECIFICITY - STABILITY INDICATING EVALUATION OF THE METHOD............................. FORCED DEGRADATION OF FINISHED PRODUCT AND STANDARD..................................1. Unstressed analysis...............................................................................................................2. Acid Hydrolysis stressed analysis..........................................................................................3. Base hydrolysis stressed analysis.........................................................................................4. Oxidation stressed analysis...................................................................................................5. Sunlight stressed analysis.....................................................................................................6. Heat of solution stressed analysis.........................................................................................7. Heat of powder stressed analysis.......................................................................................... System Suitability stressed analysis.......................................................................................... Placebo...................................................................................................................................... STABILITY OF STANDARD AND SAMPLE SOLUTIONS......................................................... Standard Solution...................................................................................................................... Sample Solutions....................................................................................................................... ROBUSTNESS.......................................................................................................................... Extraction................................................................................................................................... Factorial Design......................................................................................................................... CONCLUSION...........................................................................................................................ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationBACKGROUNDTherapeutically, Fluoxetine hydrochloride is a classified as a selective serotonin-reuptake inhibitor. Effectively used for the treatment of various depressions. Fluoxetine hydrochloride has been shown to have comparable efficacy to tricyclic antidepressants but with fewer anticholinergic side effects. The patent expiry becomes effective in 2001 (US). INTRODUCTIONFluoxetine capsules were prepared in two dosage strengths: 10mg and 20mg dosage strengths with the same capsule weight. The formulas are essentially similar and geometrically equivalent with the same ingredients and proportions. Minor changes in non-active proportions account for the change in active ingredient amounts from the 10 and 20 mg strength.The following validation, for the method SI-IAG-206-02 , includes assay and determination of Meta-Fluoxetine by HPLC, is based on the analytical method validation SI-IAG-209-06. Currently the method is the in-house method performed for Stability Studies. The Validation was performed on the 20mg dosage samples, IAG-21-001 and IAG-21-002.In the forced degradation studies, the two placebo samples were also used. PRECISIONSYSTEM REPEATABILITYFive replicate injections of the standard solution at the concentration of 0.4242mg/mL as described in method SI-IAG-206-02 were made and the relative standard deviation (RSD) of the peak areas was calculated.SAMPLE PEAK AREA#15390#25406#35405#45405#55406Average5402.7SD 6.1% RSD0.1ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::PRECISION - Method RepeatabilityThe full HPLC method as described in SI-IAG-206-02 was carried-out on the finished product IAG-21-001 for the 20mg dosage form. The method repeated six times and the relative standard deviation (RSD) was calculated.SAMPLENumber%ASSAYof labeled amountI 96.9II 97.8III 98.2IV 97.4V 97.7VI 98.5(%) Average97.7SD 0.6(%) RSD0.6PRECISION - Intermediate PrecisionThe full method as described in SI-IAG-206-02 was carried-out on the finished product IAG-21-001 for the 20mg dosage form. The method was repeated six times by a second analyst on a different day using a different HPLC instrument. The average assay and the relative standard deviation (RSD) were calculated.SAMPLENumber% ASSAYof labeled amountI 98.3II 96.3III 94.6IV 96.3V 97.8VI 93.3Average (%)96.1SD 2.0RSD (%)2.1The difference between the average results of method repeatability and the intermediate precision is 1.7%.HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationLINEARITYStandard solutions were prepared at 50% to 200% of the nominal concentration required by the assay procedure. Linear regression analysis demonstrated acceptability of the method for quantitative analysis over the concentration range required. Y-Intercept was found to be insignificant.RANGEDifferent concentrations of the sample (IAG-21-001) for the 20mg dosage form were prepared, covering between 50% - 200% of the nominal weight of the sample.Conc. (%)Conc. (mg/mL)Peak Area% Assayof labeled amount500.20116235096.7700.27935334099.21000.39734463296.61500.64480757797.52000.79448939497.9(%) Average97.6SD 1.0(%) RSD 1.0ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::RANGE (cont.)The results demonstrate linearity as well over the specified range.Correlation coefficient (RSQ)0.99981 Slope11808.3Y -Interceptresponse at 100%* 100 (%) 0.3%ACCURACYACCURACY OF STANDARD INJECTIONSFive (5) replicate injections of the working standard solution at concentration of 0.4242mg/mL, as described in method SI-IAG-206-02 were made.INJECTIONNO.PEAK AREA%ACCURACYI 539299.7II 540599.9III 540499.9IV 5406100.0V 5407100.0Average 5402.899.9%SD 6.10.1RSD, (%)0.10.1The percent deviation from the true value wasdetermined from the linear regression lineHPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::ACCURACY OF THE DRUG PRODUCTAdmixtures of non-actives (placebo, batch IAG-21-001 ) with Fluoxetine HCl were prepared at the same proportion as in a capsule (70%-180% of the nominal concentration).Three preparations were made for each concentration and the recovery was calculated.Conc.(%)Placebo Wt.(mg)Fluoxetine HCl Wt.(mg)Peak Area%Accuracy Average (%)70%7079.477.843465102.27079.687.873427100.77079.618.013465100.0101.0100%10079.6211.25476397.910080.8011.42491799.610079.6011.42485498.398.6130%13079.7214.90640599.413080.3114.75632899.213081.3314.766402100.399.618079.9920.10863699.318079.3820.45879499.418080.0820.32874899.599.4Placebo, Batch Lot IAG-21-001HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::VALIDATION OF FLUOXETINE HClAT LOW CONCENTRATIONLINEARITY AT LOW CONCENTRATIONSStandard solution of Fluoxetine were prepared at approximately 0.02%-1.0% of the working concentration required by the method SI-IAG-206-02. Linear regression analysis demonstrated acceptability of the method for quantitative analysis over this range.ACCURACY OF FLUOXETINE HCl AT LOW CONCENTRATIONThe peak areas of the standard solution at the working concentration were measured and the percent deviation from the true value, as determined from the linear regression was calculated.SAMPLECONC.µg/100mLAREA FOUND%ACCURACYI 470.56258499.7II 470.56359098.1III 470.561585101.3IV 470.561940100.7V 470.56252599.8VI 470.56271599.5(%) AverageSlope = 132.7395299.9SD Y-Intercept = -65.872371.1(%) RSD1.1HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationSystem RepeatabilitySix replicate injections of standard solution at 0.02% and 0.05% of working concentration as described in method SI-IAG-206-02 were made and the relative standard deviation was calculated.SAMPLE FLUOXETINE HCl AREA0.02%0.05%I10173623II11503731III10103475IV10623390V10393315VI10953235Average10623462RSD, (%) 5.0 5.4Quantitation Limit - QLThe quantitation limit ( QL) was established by determining the minimum level at which the analyte was quantified. The quantitation limit for Fluoxetine HCl is 0.02% of the working standard concentration with resulting RSD (for six injections) of 5.0%. Detection Limit - DLThe detection limit (DL) was established by determining the minimum level at which the analyte was reliably detected. The detection limit of Fluoxetine HCl is about 0.01% of the working standard concentration.ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::VALIDATION FOR META-FLUOXETINE HCl(EVALUATING POSSIBLE IMPURITIES)Meta-Fluoxetine HCl linearity at 0.05% - 1.0%Relative Response Factor (F)Relative response factor for Meta-Fluoxetine HCl was determined as slope of Fluoxetine HCl divided by the slope of Meta-Fluoxetine HCl from the linearity graphs (analysed at the same time).F =132.7395274.859534= 1.8Detection Limit (DL) for Fluoxetine HClThe detection limit (DL) was established by determining the minimum level at which the analyte was reliably detected.Detection limit for Meta Fluoxetine HCl is about 0.02%.Quantitation Limit (QL) for Meta-Fluoxetine HClThe QL is determined by the analysis of samples with known concentration of Meta-Fluoxetine HCl and by establishing the minimum level at which the Meta-Fluoxetine HCl can be quantified with acceptable accuracy and precision.Six individual preparations of standard and placebo spiked with Meta-Fluoxetine HCl solution to give solution with 0.05% of Meta Fluoxetine HCl, were injected into the HPLC and the recovery was calculated.HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::META-FLUOXETINE HCl[RECOVERY IN SPIKED SAMPLES].Approx.Conc.(%)Known Conc.(µg/100ml)Area in SpikedSampleFound Conc.(µg/100mL)Recovery (%)0.0521.783326125.735118.10.0521.783326825.821118.50.0521.783292021.55799.00.0521.783324125.490117.00.0521.783287220.96996.30.0521.783328526.030119.5(%) AVERAGE111.4SD The recovery result of 6 samples is between 80%-120%.10.7(%) RSDQL for Meta Fluoxetine HCl is 0.05%.9.6Accuracy for Meta Fluoxetine HClDetermination of Accuracy for Meta-Fluoxetine HCl impurity was assessed using triplicate samples (of the drug product) spiked with known quantities of Meta Fluoxetine HCl impurity at three concentrations levels (namely 80%, 100% and 120% of the specified limit - 0.05%).The results are within specifications:For 0.4% and 0.5% recovery of 85% -115%For 0.6% recovery of 90%-110%HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::META-FLUOXETINE HCl[RECOVERY IN SPIKED SAMPLES]Approx.Conc.(%)Known Conc.(µg/100mL)Area in spikedSample Found Conc.(µg/100mL)Recovery (%)[0.4%]0.4174.2614283182.66104.820.4174.2614606187.11107.370.4174.2614351183.59105.36[0.5%]0.5217.8317344224.85103.220.5217.8316713216.1599.230.5217.8317341224.81103.20[0.6%]0.6261.3918367238.9591.420.6261.3920606269.81103.220.6261.3920237264.73101.28RECOVERY DATA DETERMINED IN SPIKED SAMPLESHPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::REPEATABILITYMethod Repeatability - Meta Fluoxetine HClThe full method (as described in SI-IAG-206-02) was carried out on the finished drug product representing lot number IAG-21-001-(1). The HPLC method repeated serially, six times and the relative standard deviation (RSD) was calculated.IAG-21-001 20mg CAPSULES - FLUOXETINESample% Meta Fluoxetine % Meta-Fluoxetine 1 in Spiked Solution10.0260.09520.0270.08630.0320.07740.0300.07450.0240.09060.0280.063AVERAGE (%)0.0280.081SD 0.0030.012RSD, (%)10.314.51NOTE :All results are less than QL (0.05%) therefore spiked samples with 0.05% Meta Fluoxetine HCl were injected.HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::Intermediate Precision - Meta-Fluoxetine HClThe full method as described in SI-IAG-206-02 was applied on the finished product IAG-21-001-(1) .It was repeated six times, with a different analyst on a different day using a different HPLC instrument.The difference between the average results obtained by the method repeatability and the intermediate precision was less than 30.0%, (11.4% for Meta-Fluoxetine HCl as is and 28.5% for spiked solution).IAG-21-001 20mg - CAPSULES FLUOXETINESample N o:Percentage Meta-fluoxetine% Meta-fluoxetine 1 in spiked solution10.0260.06920.0270.05730.0120.06140.0210.05850.0360.05560.0270.079(%) AVERAGE0.0250.063SD 0.0080.009(%) RSD31.514.51NOTE:All results obtained were well below the QL (0.05%) thus spiked samples slightly greater than 0.05% Meta-Fluoxetine HCl were injected. The RSD at the QL of the spiked solution was 14.5%HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationSPECIFICITY - STABILITY INDICATING EVALUATIONDemonstration of the Stability Indicating parameters of the HPLC assay method [SI-IAG-206-02] for Fluoxetine 10 & 20mg capsules, a suitable photo-diode array detector was incorporated utilizing a commercial chromatography software managing system2, and applied to analyze a range of stressed samples of the finished drug product.GLOSSARY of PEAK PURITY RESULT NOTATION (as reported2):Purity Angle-is a measure of spectral non-homogeneity across a peak, i.e. the weighed average of all spectral contrast angles calculated by comparing all spectra in the integrated peak against the peak apex spectrum.Purity Threshold-is the sum of noise angle3 and solvent angle4. It is the limit of detection of shape differences between two spectra.Match Angle-is a comparison of the spectrum at the peak apex against a library spectrum.Match Threshold-is the sum of the match noise angle3 and match solvent angle4.3Noise Angle-is a measure of spectral non-homogeneity caused by system noise.4Solvent Angle-is a measure of spectral non-homogeneity caused by solvent composition.OVERVIEWT he assay of the main peak in each stressed solution is calculated according to the assay method SI-IAG-206-02, against the Standard Solution, injected on the same day.I f the Purity Angle is smaller than the Purity Threshold and the Match Angle is smaller than the Match Threshold, no significant differences between spectra can be detected. As a result no spectroscopic evidence for co-elution is evident and the peak is considered to be pure.T he stressed condition study indicated that the Fluoxetine peak is free from any appreciable degradation interference under the stressed conditions tested. Observed degradation products peaks were well separated from the main peak.1® PDA-996 Waters™ ; 2[Millennium 2010]ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationFORCED DEGRADATION OF FINISHED PRODUCT & STANDARD 1.UNSTRESSED SAMPLE1.1.Sample IAG-21-001 (2) (20mg/capsule) was prepared as stated in SI-IAG-206-02 and injected into the HPLC system. The calculated assay is 98.5%.SAMPLE - UNSTRESSEDFluoxetine:Purity Angle:0.075Match Angle:0.407Purity Threshold:0.142Match Threshold:0.4251.2.Standard solution was prepared as stated in method SI-IAG-206-02 and injected into the HPLC system. The calculated assay is 100.0%.Fluoxetine:Purity Angle:0.078Match Angle:0.379Purity Threshold:0.146Match Threshold:0.4272.ACID HYDROLYSIS2.1.Sample solution of IAG-21-001 (2) (20mg/capsule) was prepared as in method SI-IAG-206-02 : An amount equivalent to 20mg Fluoxetine was weighed into a 50mL volumetric flask. 20mL Diluent was added and the solution sonicated for 10 minutes. 1mL of conc. HCl was added to this solution The solution was allowed to stand for 18 hours, then adjusted to about pH = 5.5 with NaOH 10N, made up to volume with Diluent and injected into the HPLC system after filtration.Fluoxetine peak intensity did NOT decrease. Assay result obtained - 98.8%.SAMPLE- ACID HYDROLYSISFluoxetine peak:Purity Angle:0.055Match Angle:0.143Purity Threshold:0.096Match Threshold:0.3712.2.Standard solution was prepared as in method SI-IAG-206-02 : about 22mg Fluoxetine HCl were weighed into a 50mL volumetric flask. 20mL Diluent were added. 2mL of conc. HCl were added to this solution. The solution was allowed to stand for 18 hours, then adjusted to about pH = 5.5 with NaOH 10N, made up to volume with Diluent and injected into the HPLC system.Fluoxetine peak intensity did NOT decrease. Assay result obtained - 97.2%.ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationSTANDARD - ACID HYDROLYSISFluoxetine peak:Purity Angle:0.060Match Angle:0.060Purity Threshold:0.099Match Threshold:0.3713.BASE HYDROLYSIS3.1.Sample solution of IAG-21-001 (2) (20mg/capsule) was prepared as per method SI-IAG-206-02 : An amount equivalent to 20mg Fluoxetine was weight into a 50mL volumetric flask. 20mL Diluent was added and the solution sonicated for 10 minutes. 1mL of 5N NaOH was added to this solution. The solution was allowed to stand for 18 hours, then adjusted to about pH = 5.5 with 5N HCl, made up to volume with Diluent and injected into the HPLC system.Fluoxetine peak intensity did NOT decrease. Assay result obtained - 99.3%.SAMPLE - BASE HYDROLYSISFluoxetine peak:Purity Angle:0.063Match Angle:0.065Purity Threshold:0.099Match Threshold:0.3623.2.Standard stock solution was prepared as per method SI-IAG-206-02 : About 22mg Fluoxetine HCl was weighed into a 50mL volumetric flask. 20mL Diluent was added. 2mL of 5N NaOH was added to this solution. The solution was allowed to stand for 18 hours, then adjusted to about pH=5.5 with 5N HCl, made up to volume with Diluent and injected into the HPLC system.Fluoxetine peak intensity did NOT decrease - 99.5%.STANDARD - BASE HYDROLYSISFluoxetine peak:Purity Angle:0.081Match Angle:0.096Purity Threshold:0.103Match Threshold:0.3634.OXIDATION4.1.Sample solution of IAG-21-001 (2) (20mg/capsule) was prepared as per method SI-IAG-206-02. An equivalent to 20mg Fluoxetine was weighed into a 50mL volumetric flask. 20mL Diluent added and the solution sonicated for 10 minutes.1.0mL of 30% H2O2 was added to the solution and allowed to stand for 5 hours, then made up to volume with Diluent, filtered and injected into HPLC system.Fluoxetine peak intensity decreased to 95.2%.ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationSAMPLE - OXIDATIONFluoxetine peak:Purity Angle:0.090Match Angle:0.400Purity Threshold:0.154Match Threshold:0.4294.2.Standard solution was prepared as in method SI-IAG-206-02 : about 22mg Fluoxetine HCl were weighed into a 50mL volumetric flask and 25mL Diluent were added. 2mL of 30% H2O2 were added to this solution which was standing for 5 hours, made up to volume with Diluent and injected into the HPLC system.Fluoxetine peak intensity decreased to 95.8%.STANDARD - OXIDATIONFluoxetine peak:Purity Angle:0.083Match Angle:0.416Purity Threshold:0.153Match Threshold:0.4295.SUNLIGHT5.1.Sample solution of IAG-21-001 (2) (20mg/capsule) was prepared as in method SI-IAG-206-02 . The solution was exposed to 500w/hr. cell sunlight for 1hour. The BST was set to 35°C and the ACT was 45°C. The vials were placed in a horizontal position (4mm vials, National + Septum were used). A Dark control solution was tested. A 2%w/v quinine solution was used as the reference absorbance solution.Fluoxetine peak decreased to 91.2% and the dark control solution showed assay of 97.0%. The difference in the absorbance in the quinine solution is 0.4227AU.Additional peak was observed at RRT of 1.5 (2.7%).The total percent of Fluoxetine peak with the degradation peak is about 93.9%.SAMPLE - SUNLIGHTFluoxetine peak:Purity Angle:0.093Match Angle:0.583Purity Threshold:0.148Match Threshold:0.825 ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationSUNLIGHT (Cont.)5.2.Working standard solution was prepared as in method SI-IAG-206-02 . The solution was exposed to 500w/hr. cell sunlight for 1.5 hour. The BST was set to 35°C and the ACT was 42°C. The vials were placed in a horizontal position (4mm vials, National + Septum were used). A Dark control solution was tested. A 2%w/v quinine solution was used as the reference absorbance solution.Fluoxetine peak was decreased to 95.2% and the dark control solution showed assay of 99.5%.The difference in the absorbance in the quinine solution is 0.4227AU.Additional peak were observed at RRT of 1.5 (2.3).The total percent of Fluoxetine peak with the degradation peak is about 97.5%. STANDARD - SUNLIGHTFluoxetine peak:Purity Angle:0.067Match Angle:0.389Purity Threshold:0.134Match Threshold:0.8196.HEAT OF SOLUTION6.1.Sample solution of IAG-21-001-(2) (20 mg/capsule) was prepared as in method SI-IAG-206-02 . Equivalent to 20mg Fluoxetine was weighed into a 50mL volumetric flask. 20mL Diluent was added and the solution was sonicated for 10 minutes and made up to volume with Diluent. 4mL solution was transferred into a suitable crucible, heated at 105°C in an oven for 2 hours. The sample was cooled to ambient temperature, filtered and injected into the HPLC system.Fluoxetine peak was decreased to 93.3%.SAMPLE - HEAT OF SOLUTION [105o C]Fluoxetine peak:Purity Angle:0.062Match Angle:0.460Purity Threshold:0.131Match Threshold:0.8186.2.Standard Working Solution (WS) was prepared under method SI-IAG-206-02 . 4mL of the working solution was transferred into a suitable crucible, placed in an oven at 105°C for 2 hours, cooled to ambient temperature and injected into the HPLC system.Fluoxetine peak intensity did not decrease - 100.5%.ED. N0: 04Effective Date:APPROVED::。

2 ＤＯＩ：１０．３９６９／ｊ．ｉｓｓｎ．１００１－５２５６．２０２３．０１．０２８细胞器之间相互作用在非酒精性脂肪性肝病发生发展中的作用刘天会首都医科大学附属北京友谊医院肝病中心，北京１０００５０通信作者：刘天会，ｌｉｕ＿ｔｉａｎｈｕｉ＠１６３．ｃｏｍ（ＯＲＣＩＤ：００００－０００１－６７８９－３０１６）摘要：细胞器除了具有各自特定的功能外，还可与其他细胞器相互作用完成重要的生理功能。

细胞器之间相互作用的异常与疾病的发生发展密切相关。

近年来，细胞器之间相互作用在非酒精性脂肪性肝病（ＮＡＦＬＤ）发生发展中的作用受到关注，特别是线粒体、脂滴与其他细胞器之间的相互作用。

关键词：非酒精性脂肪性肝病；细胞器；线粒体；脂肪滴基金项目：国家自然科学基金面上项目（８２０７０６１８）ＲｏｌｅｏｆｏｒｇａｎｅｌｌｅｉｎｔｅｒａｃｔｉｏｎｉｎｔｈｅｄｅｖｅｌｏｐｍｅｎｔａｎｄｐｒｏｇｒｅｓｓｉｏｎｏｆｎｏｎａｌｃｏｈｏｌｉｃｆａｔｔｙｌｉｖｅｒｄｉｓｅａｓｅＬＩＵＴｉａｎｈｕｉ．（ＬｉｖｅｒＲｅｓｅａｒｃｈＣｅｎｔｅｒ，ＢｅｉｊｉｎｇＦｒｉｅｎｄｓｈｉｐＨｏｓｐｉｔａｌ，ＣａｐｉｔａｌＭｅｄｉｃａｌＵｎｉｖｅｒｓｉｔｙ，Ｂｅｉｊｉｎｇ１０００５０，Ｃｈｉｎａ）Ｃｏｒｒｅｓｐｏｎｄｉｎｇａｕｔｈｏｒ：ＬＩＵＴｉａｎｈｕｉ，ｌｉｕ＿ｔｉａｎｈｕｉ＠１６３．ｃｏｍ（ＯＲＣＩＤ：００００－０００１－６７８９－３０１６）Ａｂｓｔｒａｃｔ：Ｉｎａｄｄｉｔｉｏｎｔｏｉｔｓｏｗｎｓｐｅｃｉｆｉｃｆｕｎｃｔｉｏｎｓ，ａｎｏｒｇａｎｅｌｌｅｃａｎａｌｓｏｉｎｔｅｒａｃｔｗｉｔｈｏｔｈｅｒｏｒｇａｎｅｌｌｅｓｔｏｃｏｍｐｌｅｔｅｉｍｐｏｒｔａｎｔｐｈｙｓｉｏｌｏｇｉｃａｌｆｕｎｃｔｉｏｎｓ．Ｔｈｅｄｉｓｏｒｄｅｒｓｏｆｏｒｇａｎｅｌｌｅｉｎｔｅｒａｃｔｉｏｎｓａｒｅｃｌｏｓｅｌｙａｓｓｏｃｉａｔｅｄｔｈｅｄｅｖｅｌｏｐｍｅｎｔａｎｄｐｒｏｇｒｅｓｓｉｏｎｏｆｖａｒｉｏｕｓｄｉｓｅａｓｅｓ．Ｉｎｒｅｃｅｎｔｙｅａｒｓ，ｔｈｅｒｏｌｅｏｆｏｒｇａｎｅｌｌｅｉｎｔｅｒａｃｔｉｏｎｓｈａｓａｔｔｒａｃｔｅｄｍｏｒｅａｔｔｅｎｔｉｏｎｉｎｔｈｅｐｒｏｇｒｅｓｓｉｏｎｏｆｎｏｎａｌｃｏｈｏｌｉｃｆａｔｔｙｌｉｖｅｒｄｉｓｅａｓｅ，ｅｓｐｅｃｉａｌｌｙｔｈｅｉｎｔｅｒａｃｔｉｏｎｓｂｅｔｗｅｅｎｍｉｔｏｃｈｏｎｄｒｉａ，ｌｉｐｉｄｄｒｏｐｌｅｔｓ，ａｎｄｏｔｈｅｒｏｒｇａｎｅｌｌｅｓ．Ｋｅｙｗｏｒｄｓ：Ｎｏｎ－ａｌｃｏｈｏｌｉｃＦａｔｔｙＬｉｖｅｒＤｉｓｅａｓｅ；Ｏｒｇａｎｅｌｌｅｓ；Ｍｉｔｏｃｈｏｎｄｒｉａ；ＬｉｐｉｄＤｒｏｐｌｅｔｓＲｅｓｅａｒｃｈｆｕｎｄｉｎｇ：ＮａｔｉｏｎａｌＮａｔｕｒａｌＳｃｉｅｎｃｅＦｏｕｎｄａｔｉｏｎｏｆＣｈｉｎａ（８２０７０６１８） 细胞器可以通过膜接触位点与其他细胞器相互作用，完成物质与信息的交换，形成互作网络［１］。

Multidrug resistance reversal activity of total alkaloid fromFritillaria thunbergii on cisplatin-resistant humanlung adenocarcinoma A549/DDP cellsLI Ze-hui1，AN Chao2，HU Kai-wen2，ZHOU Ke-hua3，DUAN Hui-hui1，TANG Min-ke1（1．Department of Pharmacology of Chinese Meteria Medica，School of Chinese Meteria Medica，Beijing University of Chinese Medicine，Beijing100102，China；2．Department of Oncology，East Hospital，Beijing University of Chinese Medicine，Beijing100078，China；3．Department of Physical Therapy，Daemen College，Amherst14226，USA）Abstract：OBJECTIVE To explore the effect of total alkaloid from Fritillaria thunbergii（TAF）on reversing multidrug resistance（MDR）of human lung adenocarcinoma A549/DDP cells．METHODS①In vitro Cytoxicity and proliferation inhibitory rate of TAF（12．5－200mg·L－1）was assessed by MTT method．TAF9mg·L－1was used in subsequent rever-sal experiments．Cyclosporine A（Cys A）1mg·L－1and tetrandrine（Tet）1mg·L－1acted as positive control group．The amount of MDR1mRNA and P-glycoprotein（P-gp）of A549/DDP cells was measured by real time polymerase chain reac-tion and Western blotting，respectively．②In vivo BALB/c nude mice were used to establish an A549/DDP tumor model．The mice were randomly divided into vehicle，DDP5mg·kg－1，TAF2mg·kg－1，DDP5mg·kg－1plus TAF0．5，1and 2mg·kg－1groups．DDP was ip given every two days and TAF was ig given once a day．Tumor volume was measured every four days and tumor mass was detected after13d．RESULTS After incubation with TAF for72h，IC50of TAF toA549and A549/DDP cells was141ʃ5and（298ʃ22）mg·L－1，respectively，and IC10of TAF to A549and A549/DDPcells was15．3ʃ1．9and（9．0ʃ1．2）mg·L－1．IC50of DDP without or with TAF9mg·L－1on A549/DDP cells was14．06ʃ3．72and（0．79ʃ0．14）mg·L－1，respectively，while there was no significant change in IC50of A549cells．TAF reversed DDP resistance of A549/DDP cells with fold-reversal17．80，which was higher than that of Cys A（10．16）and Tet（14．05）．Compared with A549/DDP cell vehicle group，MDR1mRNA and P-gp expression in A549/DDP cells was decreased by TAF（P＜0．01）．The tumor inhibitory rate of DDP5mg·kg－1in vivo was49．9%．The combination of DDP 5mg·kg－1and TAF2mg·kg－1increased the tumor inhibition rate to67．4%．CONCLUSION TAF enhances MDR rever-sal effect of DDP on A549/DDP cells in vitro and in vivo，and down-regulates MDR1mRNA and P-gp expression in A549/ DDP cells．Key words：total alkaloid；Fritillaria thunbergii；multidrug resistance；P-glycoproteinCLC number：R285Document code：A Article ID：1000-3002（2013）03-0315-06DOI：10．3867/j．issn．1000-3002．2013．03．002Cisplatin（DDP），a broad spectrum antitu-mor agent，is the most frequently used chemo-therapy for human lung adenocarcinoma．Howev-er，patients with lung adenocarcinoma usually re-quire long-term DDP management which is often accompanied by multidrug resistance（MDR），a challenge for patients and clinicians alike under advanced cancer conditions．Over expression of P-glycoprotein（P-gp）is one of the major mecha-nisms of MDR，as P-gp can pump drugs outside the cells and thus compromise the efficacy of Foundation item：The project supported by National Science and Technology Mega-project of China（2009ZX 09103-346）Biography：LI Ze-hui（1988－），female，graduate student，research field is Chinese medicine pharmacology．Corresponding author：HU Kai-wen，Tel：（010）67689787，E-mail：kaiwenh@163．com；TANG Min-ke，E-mail：tangmk@bucm．edu．cn，Tel：（010）64287660chemotherapeutic drugs［1］．Therefore，an effec-tive MDR reversal agent is usually required to im-prove the effects of chemotherapeutic drugs．In recent years，some Chinese herbs，such as ligustrazine［2］and tetrandrine（Tet）［3］，have been proved to have MDR reversal effects．A study by Li et al［4］found that Fritillaria thunbergii pulvis could effectively reverse MDR in acute leu-kemia．However，little information is available re-garding its effects in MDR reversal on human lung adenocarcinoma．This study was intended to in-vestigate the MDR reversal effect of total alkaloid of Fritillaria thunbergii（TAF），a type of isosteroi-dal alkaloids extracted from Fritillaria thunbergii，on human lung adenocarcinoma A549/DDP cells．1MATERIALS AND METHODS1．1Cells，reagents and drugsRPMI1640was purchased from Invitrogen（Carlsbad，USA）．Fetal bovine serums（FBS）and penicillin-streptomycin solution were pur-chased from Hyclone（Beijing，China）．Trypsin was purchased from Amresco（Dallas，USA）．Dimethyl sulfoxide（DMSO），4-methyl thiazolyl tetrazolium（MTT），trypanblue，DDP，EDTA，cyclosporine A（Cys A），Tet，and sodium car-boxymethyl cellulose（CMC-Na）were purchased from Sigma-Aldrich（St．Louis，USA）．M-MLV reverse transcriptase kit was purchased from TaKaRa（Dalian，China）．Rabbit anti-mouse P-gp polyclonal antibody was purchased from Abcam（Cambridge，England）．Mouse anti-mouse GAPDH monoclonal antibody was pur-chased from Kangcheng Biological Engineering （Shanghai，China）．Goat anti-mouse IgG（H+L）/ HRP was purchased from Zhongshanjinqiao （Beijing，China）．TAF was extracted from Bulbus F．Thunbergii which was obtained from Beijing Tongrentang （Bozhou）Yinpian Co．，Ltd．The powder was ex-tracted with2%hydrochloric acid solution twice，1h each time．The combined extracts were con-centrated and centrifuged．The pH of the collected supernatants was adjusted to9．0by10%NaOH．Then the supernatants were extracted4times with methylene chloride．The collected methylene chloride portions were evaporated to dryness as the final extracts．The total alkaloid concentration was＞60%and the main components were pei-mine and peiminine which were detected by HPLC-ELSD method．Used for cell culture，TAF was dissolved with1mol·L－1HCl solution，and then pH was adjusted to6．3．For animal adminis-tration，TAF was dissolved with0．5%CMC-Na．1．2Animal and cellsBALB/c nude mice，weighing14－16g，were provided by National Institute for Control of Pharmaceutical and Biological Products（Beijing，China）．The certificate number was SCXK（Jing）2009-0017．All procedures of the animal experi-ment and husbandry were carried out in compli-ance with national regulations and were approved by the Animal Care and Use Committee at the Cancer Institute and Hospital，the Chinese Acade-my of Medical Sciences．Human lung adenocarcinoma cells，parental cells A549，and resistant cells A549/DDP，were purchased from the Chinese Academy of Medical Sciences Tumor Cell Bank．The cells were cul-tured in RPMI1640with10%FBS，penicillin1．0ˑ105U·L－1，and streptomycin0．1g·L－1in a humidified incubator with5%CO2at37ħ．1．3Detection of cytotoxicity of TAF by MTT assayA549and A549/DDP cells were seeded into a96-well plate（4000cells per well）in RPMI 1640with10%FBS，respectively．RPMI1640 medium without cells were used as blank group．After24h，TAF12．5，25，50，100，200mg·L－1 and vehicle were separately added in the medi-um．After72h of incubation，20μl MTT solution （5g·L－1）was added into each well．The plate was incubated for another4h before the medium was discarded．DMSO150μl was added into each well and oscillated for10min to dissolve the formazan crystals．The absorbance value（A）was determined at540nm with a microplate spec-trophotometer．The inhibitory rate（%）=〔1－（ADrug－ABlank）/（AVehicle－ABlank）〕ˑ100%．50%inhibitory concentration（IC50）value and10%in-hibitory concentration（IC10）value of TAF werecalculated．IC10of TAF was used as a safe con-centration for subsequent experiments．1．4Measurement of MDR reversal effect of TAF on A549/DDP cellsA549and A549/DDP cells were seeded as mentioned above．TAF9mg·L－1，Cys A（the fi-nal concentration was1mg·L－1）and Tet（the fi-nal concentration was1mg·L－1）were added into the medium with DDP（the final concentration was 0．01，0．1，1，10and100mg·L－1）．After72h incubation，cell viability was evaluated by MTTassay．The inhibitory rate and IC50of DDP were calculated as mentioned above．Fold-resistance=（IC50of DDP to A549/DDP cells）/（IC50of DDPto A549cells）．Fold-reversal=（IC50of DDP toA549/DDP cells in DDP group/IC50of DDP to A549/DDP cells in DDP plus TAF group）．1．5Detection of MDR1mRNA expression by real time PCR（RT-PCR）A549and A549/DDP cells were adjusted to 2ˑ107L－1and seeded into25cm2culture flasks．After treatment with drugs for72h，cells were col-lected．Total RNA was isolated from cultured cells using1ml Trizol reagent．The amount of total RNA was3μg and the total volume was25μl．The procedures for reverse transcription were 42ħfor60min and70ħfor10min．The forward primer was5'-AGGTTCTGGGAAGATCGCTA-3'，and the reverse primer was5'-ATACATCATTGC-CTGGGTGA-3'．PCR amplification was performed with the initial denaturation at94ħfor15min，then40cycles at94ħfor15s，60ħfor34s，72ħfor15s，followed by a final extension at 72ħfor15min．Sample MDR1cycle threshold （Ct）was normalized relative to sample reference gene GAPDH．The expression level of MDR1 gene was expressed by2－ΔCt（ΔCt=CtMDR1－CtGAPDH，Ct was the point at which the fluores-cence crossed the threshold）．1．6Determination of P-gp expression by Western blottingCells were lysed with the lysis buffer 〔NaCl150mmol·L－1，Tris-HCl50mmol·L－1（pH8．0），0．5%deoxycholic acid，0．025%NaN3，0．1%SDS，PMSF（α-toluenesulphonyl fluoride）0．1g·L－1，aprotinin1mg·L－1，0．1%NP-40〕．Fortyμg protein of whole lysate was subjected to sodium dodecylsulfate-polyacrylamide gel electro-phoresis．After the protein was transferred to pol-yvinylidene fluoride membranes and blocked at room temperature for60min，the membranes were incubated with rabbit anti-mouse P-gp poly-clonal antibody at4ħovernight．The membrane was incubated with goat anti-mouse IgG antibody at room temperature for60min．The protein was visualized with manual X-ray．The level of P-pg was calculated using the density ratio of P-pg toGAPDH calculated（IAP-pg /IAGAPDH）．1．7Determination of tumor inhibitory rate in vivo A549/DDP cells were collected and diluted with RPMI1640medium to5ˑ1010L－1and then 0．2ml was inoculated in the right front armpits of each mouse．When the tumor size reached a di-ameter of0．5cm（7d after inoculated），the mice were randomized into vehicle（0．5%CMC-Na），DDP5mg·kg－1，TAF2mg·kg－1，DDP5mg·kg－1+ TAF0．5，1and2mg·kg－1groups（ten mice per group）．DDP was ip given every two days and TAF was ig given once daily，and the tumor vol-ume was measured every four days．After being administrated for13d，all mice were sacrificed and tumors were excised and weighed．Tumor volume（TV）［5］=b2ˑl/2．Tumor inhibitory rate （%）=（W－Wt）ˑ100%（W is the average tumor weight of the vehicle group，and Wt is the average tumor weight of individual treatment group［6］）．1．8Statistical analysisStatistical analysis was performed using SPSS version17．0，and statistical differenceswere analyzed by one-way analysis of variance （ANOVA）．Results were presented as xʃs．The probit analysis method was used to determine the IC50values and95%confidence intervals．Differ-ences were considered significant if P＜0．05．2RESULTS2．1Cytotoxicity of TAF on A549and A549/DDP cellsAfter incubated with TAF for72h，IC50of TAF to A549and A549/DDP were141ʃ5and（298ʃ22）mg·L－1，respectively．IC10of TAF to A549and A549/DDP was15．3ʃ1．9and （9．0ʃ1．2）mg·L－1．The concentration at9mg·L－1 of TAF was used for the following reversal experi-ments．2．2MDR reversal effect of TAF on A549/DDPThe IC50of DDP for A549and A549/DDP cells was0．54ʃ0．11and（14．06ʃ0．89）mg·L－1．The multiple of drug resistance was26．03．After the cells were treated with DDP plus TAF9mg·L－1，IC50of DDP to A549and A549/DDP cells was 1．93ʃ0．12and（0．79ʃ0．06）mg·L－1，respec-tively．TAF9mg·L－1did not significantly enhance the inhibition effect of DDP on A549cells（Fig．1A）．However，it could significantly improve the effect of DDP on A549/DDP cells（P＜0．01）（Fig．1B）．The fold of MDR of A549/DDP cells was decreased to0．41．TAF，Cys A，and Tet reversed DDP resist-ance of A549/DDP cells with fold-reversal17．80，10．16and14．05，respectively．The results indi-cated that TAF could partly reverse the resistance of A549/DDP cells to DDP（Fig．1B）．2．3Effect of TAF on MDR1mRNA expression of A549/DDP cellsFig．2suggested that A549/DDP cells ex-press a higher level of MDR1mRNA than A549cells （P＜0．01）．TAF9mg·L－1or DDP14mg·L－1used alone significantly down-regulated the relative lev-el of MDR1mRNA on A549/DDP cells，com-pared with A549/DDP cell vehicle group（P＜0．01）．However，the relative level of MDR1 mRNA in DDP14mg·L－1group and DDP14mg·L－1 plus TAF9mg·L－1group did not show significant difference．2．4Effect of TAF on P-glycoprotein expres-sion of A549/DDP cellsCompared with A549cells，the density ratioFig．1Multidrug resistance （MDR ）reversal effect of Fritillaria thunbergii （TAF ）on A549/DDP cells．A549（A ）and A549/DDP （B ）cells were exposed to cisplatin （DDP ），DDP plus TAF ，DDP plus cyclosporine A （Cys A ），and DDP plus tetrandrine （Tet ）for 72h ，respectively．Cell viability was deter-mined by MTT assay．TAF ，Cys A ，and Tet reversed DDP resist-ance of A549/DDP cells with fold-reversal 17．80，10．16and 14．05，respectively．x ʃs ，n =3．Fig．2Effect of TAF on MDR1mRNA expression detected by real time PCR．A549/DDP cells and A549cellswere treated with drugs for 72h．1：A549/DDP cells ，vehicle ；2：A549/DDP cells ，TAF 9mg ·L －1；3：A549/DDP cells ，DDP 14mg ·L －1；4：A549/DDP cells ，TAF 9mg ·L －1plus DDP14mg ·L －1；5：A549cells ，vehicle．x ʃs ，n =3．＊＊P ＜0．01，compared with A549vehicle group ；##P ＜0．01，compared with A549/DDP vehicle group ；△△P ＜0．01，compared with TAF9mg·L －1group．of P-pg protein on A549/DDP cells was remark-ably increased （P ＜0．01）．TAF 9mg ·L －1or DDP 14mg ·L －1used alone could down-regulatethe relative level of P-gp on A549/DDP cells．However ，the relative level of P-gp in DDP 14mg ·L －1plus TAF 9mg ·L －1group did not show significant difference compared with DDP 14mg ·L －1group （Fig．3）．Fig．3Effect of TAF on P-glycoprotein （P-gp ）expres-sion detected by Western blotting．See Fig．2for cell treat-ment．1：A549/DDP cells ，vehicle ；2：A549/DDP cells ，TAF 9mg ·L －1；3：A549/DDP cells ，DDP 14mg ·L －1；4：A549/DDPcells ，TAF 9mg·L －1plus DDP 14mg ·L －1；5：A549cells ，vehi-cle．IA ：integrated absorbance．x ʃs ，n =3．＊＊P ＜0．01，com-pared with A549vehicle group ；##P ＜0．01，compared with A549/DDP vehicle group ；△△P ＜0．01，compared with TAF 9mg ·L －1group．2．5Effect of TAF on A549/DDP transplant-able tumor growth in vivoTAF alone did not show significant tumor in-hibitory effect on A549/DDP transplantation tumor growth．DDP 5mg ·kg －1inhibited tumor growth significantly．The combination of DDP 5mg ·kg －1and TAF 2mg ·kg －1decreased the tumor volume and mass ，compared with DDP 5mg ·kg －1group （P ＜0．01）．The tumor inhibitory rate of DDP was remarkably increased from 49．9%to 67．4%after treatment with DDP plus TAF 2mg ·kg －1（Tab．1），showing that TAF enhanced the tumor inhibi-tion effect of DDP．Tab．1Effect of TAF on A549/DDP transplantable tumor growth in vivoGroup Tumor volume/mm3Tumor mass/g Tumor inhibition rate/% Model control772ʃ5860．77ʃ0．060．0DDP5mg·kg－1403ʃ156＊＊0．38ʃ0．04＊＊49．9TAF2mg·kg－1653ʃ4820．66ʃ0．0614．3DDP+TAF0．5mg·kg－1388ʃ210＊＊0．38ʃ0．07＊＊50．5 1mg·kg－1308ʃ128＊＊0．31ʃ0．05＊＊60．22mg·kg－1288ʃ161＊＊##0．25ʃ0．04＊＊##67．4A549/DDP cells（5ˑ1010L－1）were inoculated in the right front armpits of the mice．Seven days after inoculation，DDP was ip given every two days and TAF was ig given once daily．After administrated for13d，all the mice were sacrificed and tumors were excised and weighed．Tumor inhibition rate（%）=（W－Wt）/Wˑ100%（W is the average tumor weight of the vehicle group，and Wt is the average tumor weight of the each treatment group）．xʃs，n=10．＊＊P＜0．01，compared with model group；##P＜0．01，compared with DDP5mg·kg－1 group．3DISCUSSIONThis study used a concentration of9mg·L－1 TAF（IC10of TAF to A549/DDP cells）to observe its MDR reversal effect．The results showed thatTAF9mg·L－1significantly decreased IC50of DDP on A549/DDP cells and decreased the drug re-sistance multiple．In vivo，TAF2mg·kg－1ip each day could enhance the suppression effect of DDP on A549/DDP tumor cells．DDP is the first-line antitumor drug in clinical treatment of lung cancer and ovarian cancer．However，many different tumors enhibit［7－9］MDR to DDP and other chemotherapeutics after the remission of syndrome．One important cause of MDR is the over-expression of P-gp，which can pump drugs to extracellular membranes．P-gp is coded by MDR1gene．The normal function of the protein is to secrete steroids and to metabolize tox-icant［10－11］．A549/DDP cells in this study ex-pressed a high level of MDR1mRNA and P-gp，which is consistent with other studies［12］．TAF 9mg·L－1showed MDR reversal activity when used together with DDP in vitro．In addition，TAF also enhanced the suppression effects of DDP on A549/DDP transplantation tumor in mice．When used alone，TAF induced a downregulation of MDR1mRNA and reduced the relative level of P-gp on A549/DDP cells．However，the level of MDR1mRNA and P-gp of A549/DDP cells in TAF 9mg·L－1plus DDP14mg·L－1group did not show significant difference with those in DDP 14mg·L－1group，suggesting that TAF may sup-press tumor growth through pathways other than regulation of MDR1mRNA and P-gp．The present study showed for the first time that TAF，an active ingredient in Fritillaria thun-bergii，could reverse the resistance of A549/DDP cells to DDP both in vitro and in vivo．Further studies are needed to explore the mechanism of TAF on A549/DDP cells．REFERENCES：［1］Bosch I，Croop J．P-glycoprotein multidrug resistance and cancer［J］．Biochim Biophys Acta，1996，1288（2）：F37-F54．［2］Mei Y，Shi YJ，Zuo GQ，Gong JP，Liu CA，Li XH，et al．Study on ligustrazine in reversing multidrug resistance ofHepG2/ADM cell in vitro［J］．China J Chin Mater Med（中国中药杂志），2004，29（10）：970-973．［3］Fu LW，Deng ZA，Pan QC，Fan W．Screening and dis-covery of novel MDR modifiers from naturally occurring bis-benzylisoquinoline alkaloids［J］．Anticancer Res，2001，21（4A）：2273-2280．［4］Li W，Hu KW，Su W，Sun YL，Chen XY，Liang B．Clinical trial of Fritillaria hunbergii bulb powder for reversing multi-drug resistance in the patients with acute leukemia［J］．JBeijing Univ Tradit Chin Med（北京中医药大学学报），2004，27（1）：63-65．［5］Zalatnai A，Molnár J．Effect of SILA-409，a new organosil-icon multidrug resistance modifier，on human pancreaticcancer xenografts［J］．In Vivo，2006，20（1）：137-140．［6］Liu Z，Ren Y，Pan L，Xu HM．In vivo anti-tumor activity of polypeptide HM-3Modified by different polyethylene glycols（PEG）［J］．Int J Mol Sci，2011，12（4）：2650-2663．［7］Tai DJ，Jin WS，Wu CS，Si HW，Cao XD，Guo AJ，et al．Changes in intracellular redox status influence multidrug re-sistance in gastric adenocarcinoma cells［J］．Exp TherMed，2012，4（2）：291-296．［8］Zhu ZA，Zhu ZQ，Cai HX，Liu Y．Reversion of multidrug resistance by SKI-Ⅱin SGC7901/DDP cells and explora-tion of underlying mechanisms［J］．Asian Pac J CancerPrev，2012，13（2）：625-631．［9］Zhou Y，Ling XL．Establishment of a cisplatin-induced mul-tidrug resistance cell line SK-Hep1/DDP［J］．Chin J Canc-er，2010，29（2）：167-171．［10］Randolph GJ，Beaulieu S，Pope M，Sugawara I，Hoffman L，Steinman RM，et al．A physiologic function for p-glyco-protein（MDR-1）during the migration of dendritic cellsfrom skin via afferent lymphatic vessels［J］．Proc NatlAcad Sci USA，1998，95（12）：6924-6929．［11］Schinkel AH，Mayer U，Wagenaar E，Mol CA，van Deemter L，Smit JJ，et al．Normal viability and alteredpharmacokinetics in mice lacking mdr1-type（drug-trans-porting）P-glycoproteins［J］．Proc Natl Acad Sci USA，1997，94（8）：4028-4033．［12］LüJ，Tian YF．Effect of Src tyrosine kinase inhibition on the drug-resistance as well as MDR1and LRP expressionof the human Cis-platinum-resistant lung cancer cell lineA549/DDP［J］．Chin J Lung Cancer（中国肺癌杂志），2012，15（9）：501-506．浙贝母总生物碱对人肺腺癌A549/顺铂细胞耐药性的逆转作用李泽慧1，安超2，胡凯文2，周科华3，段惠惠1，唐民科1（1．北京中医药大学中药学院中药药理系，北京100102；2．北京中医药大学东方医院肿瘤科，北京100078；3．Department of Physical Therapy，Daemen College，Amherst14226，USA）摘要：目的研究浙贝母总生物碱（TAF）对人肺腺癌A549/顺铂（DDP）细胞DDP耐药性的逆转作用。

2018•08科研开发当代化工研究Chenmical I ntermediate丄〇丄以4-异丁基苯甲酸为原料合成布洛芬的研究*薛文凤刘浩(西北民族大学化工学院甘肃730124)搞要：布洛芬作为一种新型的非甾体类的镇痛、消炎和解热类药物，其因与传统的阿司匹林药物相比较，因其药效作用更强，副作用更 小，而被成为“超级阿司匹林”。

并引起了人们的广泛关注，现今该药物在非甾体类药物当中，销售量较大。

所以研究该用药物的合成，将会具有十分重大的意义•鉴于此，本文研究了以4-异丁基苯曱醛为原料合成布洛芬。

关键词：布洛芬；合成；4-异丁基苯曱醛；原料；工艺中图分类号：0文献标识码：AStudy on Synthesis of Ibuprofen Taking 4-isobutyl Benzaldehyde as the Raw MaterialXue Wenfeng,Liu Hao(Chemistry Institute of Northwestern Nationalities University,Gansu,730124) Abstract: Ibuprofen is a new type o f n on-steroidal analgesic, anti-inflam m atory and antipyretic drug. Compared w ith tra d itio n a l asp irin, it is called "super a sp irin” because o f i ts stronger drug effect and s m aller side effects, and it has attractedpeople's extensive attention. Among non-steroidal drugs, ibuprofen has a la rge r sales volume. Therefore, it w ill be o fg re a t s ignificance to study the synthesisof t his drug. In view o f t his, th is p aper studiedthe synthesis o f i buprofen taking 4 - isobutyl benzaldehyde as the raw m aterial.Key wordsi i b uprofen% synthesis\ 4-isobutyl benzaldehyde% raw m aterials\ process布洛芬（ibuprofen,简称为IB),化学名称为异丁基 丙稀酸或2-(4-异丁基苯基)丙酸，中文名称为拔怒风，是当前世界上一种比较热销的用于抗炎、解热镇痛类的非处方药 物，属于一种比较典型的，新一代非甾体类的抗炎药物，该药物为经典解热镇痛药物，作为阿司匹林的替代品，但是其 与阿司匹林药物相比较，其消炎、解热和镇痛的效果更好，而且其副作用相对于阿司匹林来说更小，常常被誉为“超级阿司匹林”。

SYNCHRON ®System(s)ALTChemistry Information Sheet©Copyright 2010Beckman Coulter,Inc.A l a n i n e A m i n o t r a n s f e r a s eREF 442620(200tests/cartridge)REF 476826(400tests/cartridge)For In Vitro Diagnostic UseANNUAL REVIEW Reviewed by:DateReviewed by:DatePRINCIPLEINTENDED USEALT reagent,when used in conjunction with SYNCHRON LX ®System(s),UniCel ®DxC 600/800System(s),is intended for the quantitative determination of alanine aminotransferase activity in human serum or plasma.CLINICAL SIGNIFICANCEAlanine aminotransferase measurements are used in the diagnosis and treatment of certain liver diseases (e.g.,viral hepatitis and cirrhosis)and heart diseases.METHODOLOGYALT reagent is used to measure analyte activity by a kinetic rate method.1,2In the reaction,alanine aminotransferase catalyzes the reversible transamination of L-alanine and alpha-ketoglutarate to pyruvate and L-glutamate.The pyruvate is then reduced to lactate in the presence of lactate dehydrogenase (LDH)with the concurrent oxidation of reduced β-nicotinamide adenine dinucleotide (NADH)to β-nicotinamide adenine dinucleotide (NAD).The SYNCHRON ®System(s)automatically proportions the appropriate sample and reagent volumes into the cuvette.The ratio used is one part sample to 11parts reagent.The system monitors the change in absorbance at 340nanometers.This change in absorbance is directly proportional to the activity of ALT in the sample and is used by the System to calculate and express the ALT activity.CHEMICAL REACTION SCHEMESPECIMENTYPE OF SPECIMENBiological fluid samples should be collected in the same manner routinely used for any laboratory test.3Freshly drawn serum or plasma are the specimens of choice.Acceptable anticoagulants are listed in PROCEDURAL NOTES section of this chemistry information sheet.Whole blood is not recommended for use as a sample.SPECIMEN STORAGE AND STABILITY1.Tubes of blood are to be kept closed at all times and in a vertical position.It is recommended that the serum orplasma be physically separated from contact with cells within two hours from the time of collection.42.Separated serum or plasma should not remain at room temperature longer than8hours.If assays are notcompleted within8hours,serum or plasma should be stored at+2°C to+8°C.If assays are not completed within 48hours,or the separated sample is to be stored beyond48hours,samples should be frozen at-15°C to-20°C.Frozen samples should be thawed only once.Analyte deterioration may occur in samples that are repeatedly frozen and thawed.43.Refer to references(4,5)for additional information on the effects of preanalytical variables on sample storage andstability.Each laboratory should determine if the recommended requirements are appropriate.Additional specimen storage and stability conditions as designated by this laboratory:SAMPLE VOLUMEThe optimum volume,when using a0.5mL sample cup,is0.3mL of sample.For optimum primary sample tube volumes and minimum volumes,refer to the Primary Tube Sample Template for your system.CRITERIA FOR UNACCEPTABLE SPECIMENSRefer to the PROCEDURAL NOTES section of this chemistry information sheet for information on unacceptable specimens.Criteria for sample rejection as designated by this laboratory:PATIENT PREPARATIONSpecial instructions for patient preparation as designated by this laboratory:SPECIMEN HANDLINGSpecial instructions for specimen handling as designated by this laboratory:REAGENTSCONTENTSEach kit contains the following items:Two Alanine Aminotransferase Reagent Cartridges(2x200tests)or(2x400tests and2bottles of ALT[A-reagent]) VOLUMES PER TESTSample Volume23µLORDAC Sample Volume3µLTotal Reagent Volume250µLCartridge VolumesA242µLB8µLC––REACTIVE INGREDIENTSREAGENT CONSTITUENTSα-Ketoglutarate16mmol/LLactate dehydrogenase(LD)>2300IU/LL-Alanine500mmol/LTris buffer97mmol/LNADH0.18mmol/LAlso non-reactive chemicals necessary for optimal system performance.Avoid skin contact with e water to wash reagent from skin.EUROPEAN HAZARD CLASSIFICATIONXn;R22Harmful if swallowed.Alanine Aminotransferase Reagent(Compartment B)S37/39Wear suitable gloves and eye/face protection. MATERIALS NEEDED BUT NOT SUPPLIED WITH REAGENT KITAt least two levels of control materialSalineREAGENT PREPARATIONFor P/N442620(200tests):Transfer all the contents of the smallest reagent compartment(C)into the largest reagent compartment(A).For P/N476826(2x400tests):Transfer all the contents of one ALT(A-reagent)bottle into the largest reagent compartment(A).Replace cartridge caps and gently invert the cartridge several times to ensure adequate mixing.ACCEPTABLE REAGENT PERFORMANCEThe acceptability of a reagent is determined by ensuring that quality control results are within your facility’s acceptance criteria.REAGENT STORAGE AND STABILITYALT reagent when stored unopened at+2°C to+8°C will obtain the shelf-life indicated on the cartridge label.Once prepared,the reagent is stable for30days at+2°C to+8°C unless the expiration date is exceeded.DO NOT FREEZE. Reagent storage location:CALIBRATIONCALIBRATOR REQUIREDCalibration is not required.TRACEABILITYThis measurand(analyte)is traceable to the manufacturer’s selected Measurement Procedure as described in the Methodology section.QUALITY CONTROLAt least two levels of control material,normal and abnormal,should be analyzed daily.In addition,these controls should be run with each new reagent cartridge and after specific maintenance or troubleshooting procedures as detailed in the appropriate system manual.More frequent use of controls or the use of additional controls is left to the discretion of the user based on good laboratory practices or laboratory accreditation requirements and applicable laws.The following controls should be prepared and used in accordance with the package inserts.Discrepant quality control results should be evaluated by your facility.Table1.0Quality Control MaterialCONTROL NAME SAMPLE TYPE STORAGETESTING PROCEDURE(S)1.If necessary prepare reagent as defined in the Reagent Preparation section of this chemistry information sheet andload the reagent onto the system.2.Program samples and controls for analysis.3.After loading samples and controls onto the system,follow the protocols for system operations.For detailed testing procedures,refer to the SYNCHRON LX Operations Manual,or the UniCel DxC600/800System Instructions For Use(IFU)manual.CALCULATIONSThe SYNCHRON®System(s)performs all calculations internally to produce the final reported result.The system will calculate the final result for sample dilutions made by the operator when the dilution factor is entered into the system during sample programming.REPORTING RESULTSEquivalency between the SYNCHRON LX and UniCel DxC600/800Systems has been established.Chemistry results between these systems are in agreement and data from representative systems may be shown.REFERENCE INTERVALSEach laboratory should establish its own reference intervals based upon its patient population.The following reference intervals were taken from literature and a study performed on SYNCHRON Systems.6Table2.0Reference intervalsINTERVALS SAMPLE TYPE CONVENTIONAL UNITS S.I.UNITSSerum or Plasma(Male)10–40IU/L0.17–0.68µkat/L LiteratureSerum or Plasma(Female)7–35IU/L0.12–0.60µkat/LSerum or Plasma(Male)17–63IU/L0.29–1.07µkat/L SYNCHRONSerum or Plasma(Female)14–54IU/L0.24–0.92µkat/L INTERVALS SAMPLE TYPE CONVENTIONAL UNITS S.I.UNITS LaboratoryRefer to References(7,8,9)for guidelines on establishing laboratory-specific reference intervals.Additional reporting information as designated by this laboratory:PROCEDURAL NOTESANTICOAGULANT TEST RESULTS1.If plasma is the sample of choice,the following anticoagulants were found to be compatible with this method basedon a study of20healthy volunteers:Table3.0Compatible AnticoagulantsANTICOAGULANT LEVEL TESTED FOR IN VITROINTERFERENCEAVERAGE PLASMA-SERUMBIAS(IU/L)Ammonium Heparin14Units/mL NSI a Lithium Heparin14Units/mL NSI Sodium Heparin14Units/mL NSIa NSI=No Significant Interference(within±6.0IU/L or7%).2.The following anticoagulant was found to be incompatible with this method:Table4.0Incompatible AnticoagulantsANTICOAGULANT LEVEL TESTED FOR IN VITROINTERFERENCE PLASMA-SERUM BIAS(IU/L)aPotassium Oxalate/SodiumFluoride2.0/2.5mg/mL-15a Bias is based on worst case instead of average.Plus(+)or minus(-)signs in this column signify positive or negative bias.LIMITATIONSSamples with extremely high enzyme activity(>12,000IU/L or>200.04µkat/L)may consume all of the NADH substrate before the first absorbance measurement is taken after sample addition.These samples can report either very low enzyme activities or suppress the result as"OIR LO".These samples should be diluted1:20with saline and rerun.INTERFERENCES1.The following substances were tested for interference with this methodology:Table5.0InterferencesSUBSTANCE SOURCE LEVEL TESTED OBSERVED EFFECT Bilirubin Bovine30mg/dL NSI aLipemia Intralipid b300mg/dL NSIa NSI=No Significant Interference(within±6.0IU/L or7%).b Intralipid is a registered trademark of KabiVitrum,Inc.,Clayton,NC27250.2.Samples showing evidence of hemolysis should not be used.Hemolysis may cause falsely elevated results.3.Refer to References(10,11,5)for other interferences caused by drugs,disease and preanalytical variables. PERFORMANCE CHARACTERISTICSANALYTIC RANGEThe SYNCHRON®System(s)method for the determination of alanine aminotransferase provides the following analytical range:Table6.0Analytical RangeSAMPLE TYPE CONVENTIONAL UNITS S.I.UNITSSerum or Plasma5–400IU/L0.09–6.80µkat/LSerum or Plasma(ORDAC)a350–2600IU/L 5.8–43.0µkat/La Overrange Detection and Correction.Refer to the SYNCHRON LX Operations Manual,or the UniCel DxC600/800System Instructions For Use(IFU)manual for more details on this function.Samples with activities exceeding the high end of the analytical range should be rerun with ORDAC enabled or diluted with saline and reanalyzed.REPORTABLE RANGE(AS DETERMINED ON SITE):Table7.0Reportable RangeSAMPLE TYPE CONVENTIONAL UNITS S.I.UNITSSENSITIVITYSensitivity is defined as the lowest measurable concentration which can be distinguished from zero with95%confidence. Sensitivity for ALT determination is5IU/L(0.08µkat/L).EQUIVALENCYEquivalency was assessed by Deming regression analysis of patient samples to accepted clinical methods.Serum or Plasma (in the range of 5.0to 389.1IU/L):Y (SYNCHRON LX Systems)=0.994X +5.02N=74MEAN (SYNCHRON LX Systems)=86.9MEAN (SYNCHRON CX7DELTA)=82.3CORRELATION COEFFICIENT (r)=0.9965Refer to References (12)for guidelines on performing equivalency testing.PRECISIONA properly operating SYNCHRON ®System(s)should exhibit precision values less than or equal to the following:Table 8.0Precision Values 1SDCHANGEOVER VALUE aTYPE OF PRECISION SAMPLE TYPE IU/L µkat/L IU/L µkat/L %CV Serum/Plasma 3.00.0585.7 1.43 3.5Within-run Serum/Plasma (ORDAC)NA b NA NA NA 10.0Serum/Plasma 4.50.0885.7 1.43 5.3TotalSerum/Plasma (ORDAC)NANANANA15.0aWhen the mean of the test precision data is less than or equal to the changeover value,compare the test SD to the SD guideline given above to determine the acceptability of the precision testing.When the mean of the test precision data is greater than the changeover value,compare the test %CV to the guideline given above to determine acceptability.Changeover value =(SD guideline/CV guideline)x 100.bNA =Not applicable.Comparative performance data for a SYNCHRON LX ®System evaluated using the NCCLS Proposed Guideline EP5-T2appears in the table below.13Each laboratory should characterize their own instrument performance for comparison purposes.Table 9.0NCCLS EP5-T2Precision Estimate MethodEP5-T2Calculated Point Estimates TYPE OF IMPRECISION SAMPLE TYPE No.SystemsNo.Data Points aTest Mean Value (IU/L)SD %CV Serum Control 118020.30.88 4.34Serum Control 2180178.90.870.48Within-runSerumControl 3180334.3 1.860.56Serum Control 118020.30.94 4.61Serum Control 2180178.9 1.250.70Total SerumControl 3180334.32.840.85aThe point estimate is based on the data from one system,run for twenty days,two runs per day,two observations per run on an instrument operated and maintained according to the manufacturer‘s instructions.NOTICEThese degrees of precision and equivalency were obtained in typical testing procedureson a SYNCHRON LX®System and are not intended to represent the performancespecifications for this reagent.ADDITIONAL INFORMATIONFor more detailed information on SYNCHRON LX Systems or UniCel DxC Systems,refer to the appropriate system manual.SHIPPING DAMAGEIf damaged product is received,notify your Beckman Coulter Clinical Support Center.REFERENCES1.Kamen,A.,Wroblewski,F.,LaDue,J.E.,J.Clin.Inv.,34:126133(1955).2.Henry,R.J.,et al.,Amer.J.Clin.Path.,34:381(1960).3.Tietz,N.W.,"Specimen Collection and Processing;Sources of Biological Variation",Textbook of ClinicalChemistry,2nd Edition,W.B.Saunders,Philadelphia,PA(1994).4.National Committee for Clinical Laboratory Standards,Procedures for the Handling and Processing of BloodSpecimens,Approved Guideline,NCCLS publication H18-A,Villanova,PA(1995).5.Young, D.S.,Effects of Preanalytical Variables on Clinical Laboratory Tests,2nd Edition,AACC Press,Washington,D.C.(1997).6.Tietz,N.W.,Clinical Guide to Laboratory Tests,3rd Edition,W.B.Saunders,Philadelphia,PA(1995).7.National Committee for Clinical Laboratory Standards,How to Define,Determine,and Utilize Reference Intervalsin the Clinical Laboratory,Approved Guideline,NCCLS publication C28-A,Villanova,PA(1995).8.Tietz,N.W.,ed.,Fundamentals of Clinical Chemistry,3rd Edition,W.B.Saunders,Philadelphia,PA(1987).9.Henry,J.B.,Clinical Diagnosis and Management by Laboratory Methods,18th Edition,W.B.Saunders Company,Philadelphia,PA(1991).10.Young,D.S.,Effects of Drugs on Clinical Laboratory Tests,4th Edition,AACC Press,Washington,D.C.(1995).11.Friedman,R.B.,Young, D.S.,Effects of Disease on Clinical Laboratory Tests,3rd Edition,AACC Press,Washington,D.C.(1997).12.National Committee for Clinical Laboratory Standards,Method Comparison and Bias Estimation Using PatientSamples,Approved Guideline,NCCLS publication EP9-A,Villanova,PA(1995).13.National Committee for Clinical Laboratory Standards,Precision Performance of Clinical Chemistry Devices,Tentative Guideline,2nd Edition,NCCLS publication EP5-T2,Villanova,PA(1992).Beckman Coulter Ireland,Inc.,Mervue Business Park,Mervue,Galway,Ireland35391774068Beckman Coulter,Inc.,250S.Kraemer Blvd.,Brea,CA92821。

实验研究CHINESE COMMUNITY DOCTORS 由于在生物医学领域的广泛应用，纳米材料已经成为目前的研究热点。

金纳米材料(纳米金)是指直径在1～100nm范围的金颗粒，具有良好的生物相容性、尺寸效应、表面效应以及独特的光学性质[1]，在工业催化、生物医药、肿瘤治疗、生物检测等领域具有广泛的应用[2-5]。

目前的研究认为纳米金的尺寸、形状、表面配体以及作用的细胞株类型等因素决定了其毒理学性质[6-8]。

但是，金纳米颗粒的毒理学机制尚未成熟，人们对于纳米金与活性分子之间的相互作用及其生物学效应知之甚少[9，10]。

针对金纳米颗粒的表面效应，本文研究了纳米金对不同种类抗氧化剂氧化进程的影响，并分析了其作用机制，为金纳米颗粒的合理应用提供了实验基础。

资料与方法试剂与仪器：氯金酸、碳酸钾、硼氢化钠购自国药集团化学试剂有限公司；抗坏血酸(AA)、表儿茶素(EC)、2，2，6，6-Tetramethylpiperidine (TEMP)、3-Carbamoyl-2，5-dihydro-2，2，5，5-tetramethyl-1H-pyrrol-1-yloxyl(CTPO)购自Sigma 公司；ESR 自旋捕获剂5，5-Dimethyl -1-pyrroline-N-oxide (DMPO)、5-tert-Butoxycarbonyl -5-meth-yl-1-pyrroline-N-oxide(BMPO)购自Dojin-do Molecular Technologies 公司；实验用水均为超纯水装置净化的3次去离子水，电阻率＞18.2MΩ·cm。

纳米颗粒的形貌使用FEI 公司的Tecnai G2Spirit BioTWIN 透射电子显微镜(TEM)表征；紫外-可见光谱使用岛津公司的UV-3600紫外光谱仪测定；活性氧自由基使用Bruker 公司的电子自旋共振光谱(ESR)检测。

金纳米颗粒的制备：分别配制1%的氯金酸溶液(溶液1)、0.2mol/L 的碳酸钾溶液(溶液2)和0.02mol/L 硼氢化钠溶液(溶液3)。

专利名称：USE OF XANTHINE OXIDASE INHIBITORS AS ANTI-ISCHAEMIC AGENTS发明人：SINGH, Surinder,LEWIS, Anthony Edward申请号：EP95939368.0申请日：19951208公开号：EP0796099A1公开日：19970924专利内容由知识产权出版社提供摘要：The use of a compound of formula (I), in which R1 and R2 which may be the same or different are N or CH, X1 and X2 which may be the same or different are hydrogen, hydroxy, or an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclyl group and Z1 and Z2 which may be the same or different are hydrogen, hydroxy, keto (=O), or an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclyl group or one of Z1 and X1 and Z2 and X2 form a second bond of a double bond at the 1,6 or 2,3 positions with the proviso that at least one of the groupingsR1Z1X1R2Z2X2 and R1X1Z2 form a hydroxamate moiety (-N(OH)C(=O)- in which R1and/or R2 is N, Z1 and/or Z2 is =O and X1 and/or X2 is OH or R1 is N, Z2 is =O and X1 is OH and B is a 5- or 6-membered ring of formula (II) or (III), in which R4, R5, R6, R7, R8, R9 and R10 which may be the same or different are CH or N with the proviso that ring B cannot contain more than 3 ring members which are nitrogen and the ring B may optionally be substituted by one or more of hydroxy, keto (=O), and an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclyl group or a salt thereof formed with a physiologically acceptable organic or inorganic acid, for the manufacture of a medicament for use as an anti-ischaemic agent.申请人：BRITISH TECHNOLOGY GROUP LIMITED地址：101 Newington Causeway London SE1 6BU GB 国籍：GB代理机构：Percy, Richard Keith, et al更多信息请下载全文后查看。

文章编号：1000-5404（2010）04-0369-03论著α-葡萄糖苷酶抑制剂Radicamine A 的初步研究刘春艳1，2，孟爱国3，詹欢2（300072天津，天津大学药物科学与技术学院药物化学教研室1；063000河北唐山，华北煤炭医学院：药学系中药学科2；附属医院检验科3）［摘要］目的探讨Radicamine A 对α-葡萄糖苷酶的抑制作用和对小肠葡萄糖吸收的影响。

方法建立α-葡萄糖苷酶抑制模型，采用α-葡萄糖苷酶抑制实验测定Radicamine A 对α-葡萄糖苷酶的抑制作用；采用体外小肠外翻肠囊模型来测定Radicamine A 对小肠葡萄糖吸收的抑制作用。

结果Radicamine A 对α-葡萄糖苷酶抑制作用呈剂量依赖性，其IC 50为2.27mg /L ，与拜糖平对α-葡萄糖苷酶抑制作用相比较无显著性差异；且Radicamine A 能明显抑制小肠对葡萄糖的吸收（P <0.01），也呈剂量依赖性，其IC 50为0.10mg /ml 。

与拜糖平对小肠葡萄糖吸收的抑制作用相比较无显著性差异（P >0.05）。

结论Radicamine A 能显著抑制小肠葡萄糖的吸收，有望成为一种治疗糖尿病的药物。

［关键词］Radicamine A ；α-葡萄糖苷酶抑制剂；抑制作用；拜糖平［中图法分类号］R965；R977.15［文献标志码］A［通信作者］刘春艳，E-mail ：tangshan2002@ Study on Radicamine A as a α-glucosidase inhibitorLiu Chunyan 1，2，Meng Aiguo 3，Zhan Huan 2（1School of Pharmaceutical Science and Technology ，Tianjin University ，Tianjin ，300072；2Department of Pharmacy ，North China Coal Medical College ，Tangshan ，Hebei Province ，063000；3Affiliated Hospital of North China Coal Medical College ，Tangshan ，Hebei Province ，063000，China ）［Abstract ］Objective To study the inhibitory effect of radicamine A on α-glucosidase and the glu-cose ’s absorption by small intestine.MethodsInhibitory effect of radicamine A was studied both in α-gluco-sidase inhibition experiments and by a valgus cyst model of small in testine in vitro .ResultsRadicamine Ainhibited α-glucosidase and glucose ’s absorption in a dose-dependent manner （P <0.01）and its IC 50value was 2.27mg /L and 0.10mg /ml ，respectively.No significant difference was observed in the inhibitory effects of acarbose and radicamine A on the glucose ’s absorption in small intestine.Conclusion Radicamine A cansignificantly inhibit the glucose absorption in small intestine and can thus be expected to become a novel drug ofdiabetes.［Key words ］Radicamine A ；α-glucosidase inhibitor ；inhibitory effect ；acarboseCorresponding author ：Liu Chun-yan ，E-mail ：tangshan2002@α-葡萄糖苷酶抑制剂是一类新型口服降血糖药物，能竞争性抑制小肠内α-葡萄糖苷酶的活性，延缓或抑制葡萄糖在肠道吸收，有效降低餐后高血糖［1］。

甲基法尼酯在甲壳动物中的生理作用及其机制的研究进展邱锡尔;朱冬发;汤洁;崔晓雨;周彦琦;柳志业【摘要】Methyl farnesoate (MF) is a sesquiterpenoid hormone , a crustacean juvenile hormone (JH) analog.MF has similar structure and functions with JH and appears to have several important roles in the regulation of physiological proces -ses , including the influence on molting , metamorphosis , osmoregulation , ovarian maturation and soon .This review high-lights the progresses of studies on the functions of MF , molecular mechanism synthesis and degradation in the crustaceans . It will help to understand the mechanism of methyl farnesoate and solve the problems of sexual precocity and maldevelop -ment in practice .%甲基法尼酯是一种类倍半萜烯激素，与昆虫的保幼激素在结构和功能上相似，是重要的内分泌调控因子。

MF与甲壳动物的蜕皮、形态建成、渗透压调节、卵巢发育等生理活动的调控密切相关。

主要就甲基法尼酯在甲壳动物中的生理功能、分子作用机制及其合成代谢等方面的研究进展进行综述，为进一步深入探讨甲基法尼酯的作用机制和解决虾蟹类养殖实践中的性早熟与亲本发育不良等难题奠定基础。