目的 建立体外大鼠肝微粒体中CYP2C9酶活性测定方法,并以此方法评价20种中药有效成分单体对大鼠CYP2C9酶的作用.方法 以双氯芬酸(Diclofenac)为探针,在实验组大鼠肝微粒体孵育体系中加入20种中药有效成分单体,在37℃水浴温孵15 min后冰乙腈终止反应,HPLC测定各孵育体系中探针药物的代谢产物4’-羟基双氯芬酸的转化率,并与对照组比较其差异来评价各有效成分对CYP2C9酶活性的作用.结果 4’-羟基双氯芬酸、双氯芬酸及其内标香豆素三者分离良好且无其他内源性物质干扰;动力学考察表明双氯芬酸在0.25 mg/mL的大鼠肝微粒体体系中孵育15 min,测得酶动力学参数Vmax为0.145 6 nmol/(min·mg),Km为8.270 μmol/L;抑制实验考察发现:黄芩素、蛇床子素、大黄素、獐牙菜苦苷、淫羊藿苷、白藜芦醇实验组中4’-羟基双氯芬酸转化率分别为(402.60±10.58)、(210.90±10.26)、(414.46±10.32)、(509.83±26.45)、(355.44±15.87)、(387.34±22.16) pmol/(min·mg),显著低于对照组[(735.80±17.27) pmol/(min·mg),P＜0.05],其他中药成分实验组与对照组比较,其转化率无统计学差异.结论 建立了中药有效成分对CYP2C9酶作用的体外大鼠肝微粒体研究方法,并初步评价了黄芩素、蛇床子素、大黄素、獐牙菜苦苷、淫羊藿苷、白藜芦醇对CYP2C9具有抑制作用,在临床联合用药中需注意其可能引起的药物-药物相互作用.

With the growing popularity of herbal and natural medicinal products, attention has turned to possible interactions between these products and pharmaceutical drugs. In this study, we examined whether astragaloside IV (AGS‐IV) could inhibit the activity of CYP1A2 in rat liver microsomes in vitro and in vivo.

Objective To develop a HPLC method for the determination of the activity of cytochrome P450 2A5(CYP2A5) in mouse microsomes with coumarin as the probe drug.Methods An waters symmetry C 18 column was used.The mobile phase was water(0.1 % acetic acid) and acetonitrile(70∶ 30).The flow rate was 1.0 mL·min-1.The column temperature was 30 ℃ and the RF wave length was E x = 340 nm,E m = 458 nm.Coumarin was incubated with mouse liver microsomes in vitro at 37 ℃ for 20 min and stopped by addition ice acetonitrile which contained internal standard substances and centrifuged(12000 r ·min-1) for 15 min,then the production 7-hydroxycoumarin was analyzed by HPLC RF.Results The lowest detection limit of 7-hydroxycoumarin was 2 nmol·L-1(S/N≥3),and the calibration curve was linear from 8 nmol· L-1to 800 nmol·L-1.The intra-day and inter-day relative standard deviations were less than 5% respectively.The method recoveries ranged from 91.3 % ~101.4%.The kinetic parameters,K m was 2.15 μmol·L-1and V max was 9.74 pmol·(min·mg protein)-1.Conclusion This method is reliable and the results can accurately indicate CYP2A5 enzyme activity,which can be used for the kinetics study of CYP2A5 in liver microsomes.

OBJECTIVE To optimize the condition where the reaction was catalyzed by GST and lay the fundation of the accurats determination of the GST activity with CDNB.METHODS The Welch Materials Ultimate TM XB C18 ODS column(250 mm×4.6 mm,5 μm),the mobile phase was acetonitrile-water(7:3) and the flow rate was 0.8 mL·min-1.Detection wavelength was 238 nm.Firstly,under the 0.05 mg·mL-1 protein density,to foster 10 min,and then check whether CDNB reacted with GSH by the GST enzyme or not;latter on,to set CDNB and CDNB,GSH as two kinds of reaction conditions,and compare them among the different protein density,foster time,substrate concentration;at last,to choose the appropriate reaction condition from CDNB and CDNB,GSH.RESULTS The retention time of CDNB was earlyer(about 6 min),and the peak shape was better,there was not any disturbance.CDNB can response with GSH by the GST enzyme.The response by CDNB was more than which by CDNB,GSH among the different protein density,foster time,substrate concentrations(P0.05).The response were linear with protein density and foster time by CDNB,GSH,which was the appropriate reaction condition.CONCLUSION When we determined the GST enzyme activity by CDNB,we had better to select CDNB,GSH together to start the response,by which we were able to determine accuratelly the GST enzyme activity and study on kinetics of GST in rat liver microsomes.

Objective To establish a HPLC-UV method for determining the concentration of 5-HT in brain tissue of rats.Methods Adopted a SHIMADZU VP-ODS C18 reversed-phase column(250×4.6 mm,5 μm).Methanol-0.01mol/L CH3COOK buffer solution containing 10 % citric acid(1:9,V/V,pH 4.00)as mobile phase,with the flow rate was 1.00 ml/min,and the detection was wavelength of 275 nm,The column temperature was 25℃.Results A excellent linearity of 5-HT concentration curve was obtained in a rang of 0.1~20 μg/ml,and the regression equation was A = 46008.89C +1886.58(r = 0.9999).The detection limit was 40 ng/ml,the lowest detection limits of 5-HT was 100 ng/ml.And the spotting recovery was within the range of 89.62%~101.6%.Intra-day and inter-day precisions were less than 3 %.Conclusion The method is accurate,reliable,simple and feasible,which is accepted to determine the concentration of 5-HT.

County Party committee,the government attaches great importance to flood control work,the establishment of County flood control and Drought Relief Headquarters,responsible for the flood control and the prevention of geological disasters in advance of each responsibility turns after the dangerous monitoring,timely report on the command line,at all levels of the organization,rescue teams to mass transfer property,it caused no casualties and property losses,for flood control work accumulated successful experience.

Objective:To develop a stable and sensitive method with p-nitrophenol as probe drug in order to evaluate the activity of UDP-glucuronosyltransferases(UGT1A6) in rat liver microsomes by the high-performance liquid chromatograph(HPLC).Methods:An Agilent Zorbax SB-C18(250 mm×4.6 mm,5 μm) column was used.The mobile phase was methanol and 20 mmol·L-1 KH2PO4 solution(50:50,V/V).The flow rate was 0.8 mL·min-1.The UV wave length was set at 254 nm and the column temperature at 30 ℃.p-Nitrophenol was incubated with rat liver microsomes in vitro at 30 ℃ and stopped by addition ice acetonitrile and centrifuged(12000 r·min-1) for 3 min and further analyzed by HPLC.Results:The retention time of P-Nitrophenol is 8.95 min.The concentration range was 0.5～100 μmol·L-1 and the regression equation was Y=5973.12X+571.58(r=0.9999).The lowest detectable limit(LOD) was 0.1 μmol·L-1(S/N≥3) and the lower limit of quantification(LLOQ) was 0.5 μmol·L-1.The Abstraction recoveries were 104.5%-105.5%.The intra-day and inter-day relative standard deviations were all less than 15%.There were no endogenous substances existing in the incubation system which interfered with the determination of the analyses of interest.For the rat liver microsomes Km was 24.63 μmol·L-1 and Vmax was 18.87 nmol·min-1·mg·protein-1.Conclusion:The method is stable and highly sensitive for determination of P-Nitrophenol in vitro and suitable for the evaluation of UGT1A6 activity in vitro.

OBJECTIVE To provide reference for exploitation and the quality evaluation of Bupleurum smithii Wolff var.parvifolium Shan et Y.Li in Qinghai province,and to determine the content of total saponins,saikoside a and saikoside d in it.METHODS The content of total saponins was determined by spectrophotometry.The content of saikoside a and saikoside d were determined by HPLC.RESULTS The content of total saponins,saikoside a and saikoside d in Bupleurum smithii Wolff var.parvifolium Shan et Y.Li were more than 2.77%,0.54%,0.14%,respectively.The content of saikoside a and saikoside d were higher than quality criteria in China Pharmacopeia.CONCLUSION The method is simple,rapid and accurate.The mothod is suitable for quality analysis of total saponins,saikoside a and saikoside d in Bupleurum smithii Wolff var.parvifolium Shan et Y.Li in Qinghai province which possess high medicinal value.

Objective To determine the effect of astragaloside Ⅳ(AGS-Ⅳ) on the activity of CYP1A2 in rats to elucidate the possibility CYP1A2-induced interactions of drugs.Methods Analysis of theophylline,and acetamidophenol(internal standard) was achieved using waters C18 column(250 mm×4.6 mm×5 μm) and a mobile phase consisted of methanol-water(17∶83,v/v) was delivered at a flow rate of 1.0 ml/min at 30 ℃ at a wavelength of 274 nm.Seven rats were divided into 2 groups by simple random sampling.Three rats were intravenously treated with AGS-Ⅳ(3 mg/kg),and the left 4 rats were intravenously administered with an equivalent volume of 0.1% DMSO vehicle once a day for a week.On the eighth day,all of the rats were intragastrically administered theophylline.The concentration of theophylline in the plasma was determined by high performance liquid chromatography(HPLC).The distinction of pharmacokinetic parameters was compared to investigate the effect of AGS-Ⅳ on the activity of CYP1A2.Results Theophylline and acetamidophenol were separated well,the detection limit of theophylline was 80 nmol/L,and the linear rang of our method was 0.2 to 50 μmol/L.The intra-day and inter-day relative standard deviations were both less than 10%,and the recoveries were more than 91.The main pharmacokinetic parameters of area under the concentration-time cure(AUC) of theophylline were higher than control groups(131.16±3.22 vs 97.51±6.40).The apparent oral total body clearance value(CL/F) of test group and control group were 0.12±0.01 and 0.17±0.02 respectively,with the former obviously lower than the latter(P<0.05).Conclusion AGS-Ⅳ exerts obvious inhibitory effect on CYP1A2 activity in rats,so its should be cautious to co-administer with drugs metabolized by CYP1A2.

Objective:To develop an HPLC-ECD method to determine norepinephrine in mouse brain.Method:The supernatant of mouse brain tissue homogenate precipitated by 70%HClO_4 was analyzed by HPLC-ECD method.A Hanbon lichrospher C_(18) column was used in the separation.The mixture of potassium acetate buffer and methanol(95:5,pH 4.5) was used as the mobile phase.The flow rate was 1.0 ml · min~(-1).The column temperature was 25℃.The detection voltage,current and frequency value was set at 0.6 V,100 nA and 0.05 Hz,respectively.Result:Norepinephrine had good linearity within the range of 0.1-25μmol· L~(-1).Intraand inter-day RSD of the method was below 7.81%and 3.61%,respectively.The recovery of norepinephrine was 75.05%.The detection limit was 17 ng ? ml~(-1).The content of norepinephrine determined in mouse tissue was(8.94±0.71 )μg ? g~(-1).Conclusion: The method is fast,simple and accurate in the determination of norepinephrine.

OBJECTIVE To establish a highly effective HPLC method for analyzing 1-chlorine-2,4-dinitrobenzene(CDNB),and to determine the activity of GST enzyme in rat liver microsomes(RLM)by use of CDNB as probe and study its enzyme kinetics.METHODS Welch Materials Ultimate TM XB C18 column(4.6 mm×250 mm,5 μm) was used,the mobile phase was acetonitrile-water(7∶3),the flow rate was 0.8 mL·min-1,and the detection wavelength was 238 nm.CDNB was incubated with 0.02 mg·mL-1 protein in RLM at 37 ℃ for 7 min and the reaction was terminated by cool acetonitrile.The reaction solution was centrifugated and the supernatant was filtered and analyzed by HPLC.The Vmax,Km and CLint were calculated by Sigma Plot.RESULTS The retention time of CDNB was about 6 min without any interference.The lowest detection limit of CDNB was 1.0 μmol·L-1,and the calibration curve was linear from 2.5 to 100.0 μmol·L-1.The intra-day and inter-day relative standard deviations were less than 10% respectively.The method recoveries ranged from 99.38% to 108%.The reaction catalyzed by GST was terminated by acetonitrile and the incubation time was 7 min.The kinetic parameters,Vmax,Km,and CLint were 85.45 nmol·min-1·(mg protein)-1,15.09 μmol·L-1,and 5.73 mL·min-1·(mg protein)-1,respectively.CONCLUSION This method is reliable and the results can accuratelly indicate GST enzyme activity,which can be used for the kinetics study of GST in RLM.

南山流域是我县探索的由生态型向生态经济型治理转型的示范流域,在治理上坚持与“新农村建设、产业培育、区域经济发展、当地群众意愿、党建联建双带”五个结合,机制上采用“政府主导、水保搭台、部门整合、全民参与”的协作机制,在治理模式上,通过项目巩固改造提升,依托当地水资源,以道路为骨架,采用“上保、中培、下开发”的生态经济开发治理模式,实现人口、资源、环境协调发展,为宁南地区流域治理起到示范带动作用,得到水利部的充分肯定.

Objective:To establish a method evaluates cytochrome p450 1A2(CYP1A2) activity using phenacetin as a probe by high-performance liquid chromatography(HPLC)-UV detection.Methods:Column for the Shimadzu Shim-Pack VP-ODS(150 mm 4.6 mm,5 μm) and mobile phase of 100 mmol·L-1 phosphate buffer(pH 4.3)-acetonitrile were used.Detection wavelength was 245 nm.Phenacetin was incubated with rat liver microsomes at 37 ℃ for 60 min and the reaction was stopped by cold methanol.The reactive liquid was centrifuged at 12000 r min-1 for 10 min.Finally,the supernatant was analyzed by HPLC.Results:Phenacetin,acetamidophenol and 3-acetamidophenol were perfectly separated.The detection limit of phenacetin was 50 nmol L-1 and the linear range of method was 0.1 μmol·L-1 to 10 μmol·L-1.The intraday and interday relative standard deviations were less than 10% respectively.The method recoveries were more than 75%.The methanol was selected as reagent to terminate the reaction catalyzed by CYP1A2 and the incubation time was 60 min.The kinetic parameters was shown that Vmax was 0.21 nmol·min-1·mg protein-1 and Km was 20.39 μmol·L-1.Conclusions:The method is steady,accurate and suitable for assaying CYP1A2 activity which can be used to evaluate the pharmacokinetics of CYP1A2 in rat liver microsomes.

Based on the ecological and economic benefit and sustainable developnent of social economy in Ruhe valley;it is the key point that combining the optimized agricultural structure with efficientl utitization of water and vegetation constructionl.It is nessesory to Increas ecological construction of soil and water conservation and,regulat irrational use of soil structure;incrtase soil efficiency;try our best to develop self-support,commodity production and economic crop for increasing farmers' income.We should link defendable renovates into using renovates to develop explore and protect soil and water resource and plant resource in order to realize better circle of ecological environment gradually.

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