银杏叶提取物对细胞色素P450酶CYP1A2实验研究

目的　研究银杏叶提取物对大鼠细胞色素P450酶CYP1A2活性的影响。方法　银杏叶提取物10, 100mg·kg-1 2个剂量,分别经1, 5, 10天灌胃处理大鼠,以未经处理者为对照组,测定CYP1A2的酶活性。同剂量灌胃处理10天后,再单次灌胃盐酸普萘洛尔10mg·kg-1,测定普萘洛尔和N-去异丙基普萘洛尔在大鼠体内的血药浓度。结果　银杏叶提取物处理后,大鼠肝微粒体的CYP1A2酶活性显著升高,且能显著降低普萘洛尔在大鼠体内的AUC和Cmax,使N-去异丙基普萘洛尔的AUC和Cmax显著升高。结论　银杏叶提取物能显著诱导大鼠CYP1A2的酶活性,并显著影响普萘洛尔及其代谢物在大鼠体内的血药浓度。故银杏叶提取物可能影响与之同时服用、经CYP1A2代谢的药物临床疗效。     

LC-MS/MS法测定甲氯噻嗪血浓度及药动学研究

目的为了获得国产甲氯噻嗪在中国人体内的代谢情况,以便指导临床合理用药,对国产的甲氯噻嗪片在健康中国男性受试者中空腹单剂量口服不同剂量时的药动学特点进行评价。方法采用LC-MS/MS法测定血浆中甲氯噻嗪的浓度并用非房室模型计算药动学参数。结果入选的10名受试者单剂量口服3种不同剂量(2.5、5.0和10mg)的甲氯噻嗪后的AUC0→t分别为:(128.40±20.88)、(257.68±37.10)和(517.27±119.17)μg·h·L~(~(-1));AUC0→∞分别为:(150.47±24.78)、(299.52±45.56)和(602.57±129.67)μg·h·L~(-1);cmax分别为(8.43±2.21)、(16.60±2.53)和(32.08±8.44)μg·L~(-1);tmax分别为(2.60±0.70)、(2.30±0.95)和(3.25±1.27)h;T1/2分别为(17.63±2.61)、(17.37±2.62)和(17.87±2.49)h;Ka分别为(1.195±0.720)、(1.352±0.626)和(1.221±1.152)h~(-1);Ke分别为(0.0344±0.105)、(0.0391±0.0103)和(0.0362±0.0081)h~(-1);Vd分别为(236.36±69.20)、(221.79±87.94)和(299.30±204.72)L;CL分别为(0.0174±0.0039)、(0.0178±0.0035)和(0.0179±0.0036)L·h~(-1)。结论AUC0→t(r=1)和cmax(r=0.9998)在2.5~10mg范围内呈良好的线性关系。tmax和T1/2经ANOVA(SPSS10.0)统计学处理3种不同剂量间差别无统计学意义。剂量校正后AUC0→t和cmax的几何均数分别是:50.69、51.04、50.39μg·h·L~(-1)和3.26、3.28、3.06μg·L~(-1),其值基本相近。     

补中益气汤的LC-MS分析及其对免疫抑制小鼠的调节作用

目的研究补中益气汤的主要化学成分及其对环磷酰胺免疫抑制小鼠的调节作用。方法采用LC-MS方法对补中益气汤进行了液相和质谱分析,通过MTT法测定补中益气汤对环磷酰胺免疫抑制小鼠的T、B淋巴细胞增殖和NK细胞活性的影响。结果补中益气汤中主要化学成分是多糖、黄酮类和皂苷类化合物,能够显著提高环磷酰胺免疫抑制小鼠的T、B淋巴细胞增殖能力和NK细胞活性。结论LC-MS方法可以发展成为复方有效成分研究中一个有效手段,用来阐明复方药理作用的物质基础。     

龙胆苦苷在人尿中的含量测定及排泄研究

目的:建立固相萃取液质联用方法测定人尿中龙胆苦苷浓度,研究人体静脉滴注注射用秦龙苦素的主要代谢途径。方法:尿样加入咖啡因内标,经固相萃取后LC-MS/MS分析。色谱条件为:Rescek C8柱(150 mm×2.1 mm,5μm),流动相为:甲醇-10 mmol/L醋酸铵缓冲液-乙腈(50∶40∶10),流速为0.2 mL/min,质谱检测采用多反应离子监测方式。12名健康受试者随机分组交叉静脉滴注80,240,400 mg药物,用本法测定各时段尿样的浓度。结果:龙胆苦苷在30~9 000 ng/mL线性良好(r=0.998 0),回收率为91.10%~96.21%,提取回收率为100.52%~103.83%,高、中、低浓度日内和日间变异系数均小于10%;24 h内3个剂量下原形药物在尿中累积排泄率分别为(76.59±10.02)%、(71.95±12.12)%、(79.76±8.54)%,累积排泄量与给药剂量呈正比,排泄高峰为0~5.5 h。结论:本方法适用于临床药物动力学研究,注射用秦龙苦素在人体内主要是以原形龙胆苦苷经肾脏排泄。     

A novel metalloprotease from Vipera lebetina venom induces human endothelial cell apoptosis.

A novel endothelial cell apoptosis inducing metalloprotease (VLAIP) was found in the snake venom of Vipera lebetina. This metalloprotease is a heterodimeric glycoprotein with molecular mass of about 106 kDa. The protease hydrolyzes azocasein, fibrinogen and oxidized insulin B-chain. The enzyme readily hydrolyzes the Aalpha-chain and more slowly Bbeta-chain of fibrinogen. VLAIP does not cleave fibrin. The complete amino acid sequences of the two different monomers of VLAIP are deduced from the nucleotide sequences of cDNAs encoding these proteins. The full-length cDNA sequences of the VLAIP-A and VLAIP-B encode open reading frames of 616 and 614 amino acids that include signal peptide, propeptide and mature metalloproteinase with disintegrin-like and cysteine-rich domains. VLAIP belongs to the metalloprotease/disintegrin family of reprolysins and has high identity with the proteins that induce apoptosis of endothelial cells. Treatment of HUVEC cells with VLAIP induces changes in the attachment of cells to the substrate and causes cell death. We demonstrated that VLAIP inhibits endothelial cell adhesion to extracellular matrix proteins: fibrinogen, fibronectin, vitronectin, collagen I, and collagen IV. The induction of apoptosis by VLAIP was shown by means of a typical DNA fragmentation pattern of apoptotic cells as well as by monitoring phosphatidylserine externalization using annexin V-FITC staining and flow cytometric analysis.     

液质联用技术研究肽类药物药动学中的样品制备技术

肽类药物的研究开发成为新生物技术药物研究开发的热点之一。然而，建立可靠、灵敏和准确的肽类药物体内分析方法是肽类药物临床前和临床药动学研究的难点。样品制备技术是建立符合药动学研究要求的定量分析方法的关键。现综述液质联用技术在肽类药物药动学研究中的样品制备技术方法的目的、要求、特点和分类。结合近年发表的文献具体阐述了固相萃取法、蛋白质沉淀法、超滤离心法、亲和层析法和部分其他方法的原理及其应用实例。     

国产格列本脲和二甲双胍复方制剂在健康志愿者的药代动力学和生物等效性

目的　研究国产复方格列本脲和盐酸二甲双胍胶囊单剂量人体的药代动力学特性和生物等效性。方法　以国产格列本脲片和进口盐酸二甲双胍片为标准参比制剂,入选的20名受试者随机交叉口服单剂量试验与参比制剂,用液相色谱-质谱法测定血浆中格列本脲和二甲双胍的浓度。结果　格列本脲的药代动力学参数分别为:AUC0-t为(603.32±196.61)和(581.70±195.01) ng.h.mL-1,Cmax为(123.46±27.03)和(128.28±3.67) ng.mL-1,tmax为(2.8±0.7)和(2.6±0.6 ) h,t1/2为(6.54±1.24)和(6.89±1.73) h。二甲双胍药代动力学参数分别为:AUC0-t为(7832.97±2603.38)和(7765.52±2870.85) ng. h.mL-1,Cmax为(1377.55±476.60)和(1367.40±551.19 )ng.mL-1,tmax为(2.7±0.9)和(2.0±0.5 ) h,t1/2为(5.72±1.24)和(5.81±1.51) h。试验制剂中格列本脲的相对生物利用度为(104.82±19.27)%,二甲双胍的相对生物利用度为(107.30±46.40)%。结论 试验制剂与参比制剂具有生物等效性。     

Ambient mass spectrometry for rapid diagnosis of psychoactive drugs overdose in an unstable patient

A 25-year-old man suffered from consciousness change was sent to our emergency department by friends who reported that they were not sure what had happened to him. Physical examination revealed bilateral pupils dilatation, lethargy, slurred speech, and ataxia. Computer-aided tomographic scan of the brain revealed no definite evidence of intracranial lesions. Routine laboratory tests revealed total physiological turmoil. Despite immediate commencement of aggressive treatment, the patient's condition deteriorated long before the traditional drug screen provided an answer for the identities of the multiple drugs overdose. It ended up with the need for cardiopulmonary resuscitation, but in vain. At the end of the tragic event, under the suggestion of a colleague, a portion of the patient's urine specimen was sent to our university esoteric laboratory for rapid analysis by means of a newly-developed thermal desorption-electrospray ionization-mass spectrometry. Ketamine, 3,4-methylenedioxymethamphetamine, and 3,4-methylenedioxyamphetamine were identified in the urine sample within 30 s. Conventional toxicological testing techniques like gas chromatography–mass spectrometry or liquid chromatography-mass spectrometry are currently used for identifying abused drugs. One concern is their time-consuming sample pretreatment which leads to relatively low efficiency in terms of turnaround time for revealing the identity of the consumed drugs particularly when the patients are severely overdosed. We learned a lesson from this case that a more efficient toxicological identification technique is essential to expedite the process of emergency care when the patients are so heavily overdosed that they are under critical life-threatening conditions.     

基于UHPLC-QE-MS分析结合网络药理学探讨小叶黄杨生物碱治疗阿尔茨海默病的作用机制

目的 鉴定小叶黄杨中生物碱类化学成分，探讨治疗阿尔茨海默病的物质基础和作用机制。方法 表征小叶黄杨生物碱成分，并利用PubChem、SwissADME、Swiss Target Prediction、Genecards和OMIM等数据库获取小叶黄杨生物碱的活性成分和对应靶点，以及阿尔茨海默病的相关靶点；运用Cytoscape3.9.0软件绘制“成分-靶点”网络图；运用String平台构建蛋白互作网络图；运用微生信在线绘图工具进行通路富集分析可视化；用AutodockTools-1.5.6软件对筛选出的核心成分做分子对接。结果 鉴定小叶黄杨生物碱成分23个，筛选有效成分19个，对应靶点216个；筛选阿尔茨海默病靶点880个，药物-疾病交集靶点49个；GO富集分析主要相关的生物过程有化学性突触传递，顺行跨突触信号，跨突触信号等；KEGG通路富集分析中神经活性配体-受体相互作用，5-羟色胺能突触，cAMP信号等为主要信号通路。分子对接显示筛选的核心靶点和核心成分均有较强结合力。结论 本研究初步证明了小叶黄杨生物碱成分可以多成分、多靶点、多通路共同作用治疗阿尔茨海默病，为进一步深入研究小叶黄杨生物碱对阿尔茨海默病作用的物质基础以及作用机制提供参考。