黏质沙雷氏菌发酵制备舍雷肽酶及基本酶学性质研究

目的 开发微生物发酵生产舍雷肽酶的工艺,并了解基本酶学特性。方法 从家蚕蛹的肠道中分离产酶菌株,单因素实验和响应面实验优化发酵培养基,采用(NH₄)₂SO₄沉淀、超滤和DEAE离子交换层析3步纯化舍雷肽酶,电泳法测定酶的相对分子质量和等电点,考察pH和温度对酶活力和稳定性的影响,并测算酶的反应动力学常数。结果 分离获得一株产舍雷肽酶的黏质沙雷氏菌LL-413菌株,摇瓶发酵产酶活力达到1 126 U·mL^-1,10-L发酵罐中发酵产酶活性达到1 505 U·mL^-1;舍雷肽酶相对分子质量和等电点分别约为52×10³和7.2;酶活最适pH为8.0,在pH 5.0~10.0之间稳定;酶活最适温度为40 ℃,在45 ℃以下稳定。以酪蛋白为底物时,米氏常数(K_m)和最大反应速度(V_max)分别为22.3 mg·mL^-1和1 355 mg·L·min^-1。结论 利用黏质沙雷氏菌LL-413菌株生产舍雷肽酶发酵单位高,酶的蛋白水解能力强、pH和热稳定性好,研究结果为该酶的生产和应用奠定了基础。     

左卡尼汀注射液中三甲胺分离提取及其离子色谱测定法研究

目的 建立分离提取左卡尼汀注射液中三甲胺含量的方法,及采用离子色谱法对三甲胺进行检测研究。方法 以L₉(3⁴)正交试验优化提取条件,采用0.25 mol·L^-1NaOH溶液创造碱性环境,40 ℃加热20 min,连接N₂流量为0.4 L·min^-1,用0.1 mol·L^-1甲磺酸溶液吸收气体,有效实现左卡尼汀注射液中三甲胺的分离提取;采用DionexIonPac^TM CS12A(4.0 mm×250 mm)色谱柱,DionexIonPac^TM CG12A(4.0 mm×50 mm)保护柱,以17 mmol·L^-1甲磺酸溶液为淋洗液,流速0.5 mL·min^-1,柱温30 ℃,电导检测。结果 三甲胺在0.30~48.64 μg·mL^-1内线性关系良好,相关系数为0.999 3,检出限为0.012 μg·mL^-1(S/N=3),定量限为0.3 μg·mL^-1(S/N=10),精密度良好,溶液置于室温24 h或2~8 ℃48 h内稳定。平均加样回收率在97.33~100.82%内,相对标准偏差(RSD)为1.07%(n=9)。结论 该方法能够有效提取分离左卡尼汀注射液中的三甲胺,离子色谱检测方法灵敏度高,回收率和重现性良好。     

基于药效团模型虚拟筛选活络丸中治疗骨关节炎的COX-2抑制成分

目的 采用药效团模型-分子对接-结合自由计算的分层虚拟筛选策略结合酶活性抑制实验,挖掘活络丸中对环氧合酶2(cyclooxygenase-2, COX-2)有抑制作用的成分。方法 使用Schrödinger 2020-4软件中PHASE模块进行COX-2小分子抑制剂药效团模型的构建,应用筛选出来的最佳药效团模型对活络丸中药化合物库进行虚拟筛选,对筛选到符合药效团模型的成分进行基于靶点结构的分子对接、结合自由能计算,选择潜在抑制成分进行酶活性测定实验,并对活性较好的小分子进行药代动力学、毒理学性质预测。结果 构建的最佳药效团模型具有两个氢键受体和两个芳环中心;体外酶活性抑制实验结果显示,筛选出的11个潜在靶向抑制成分均对COX-2具有不同程度的抑制作用,其中抑制活性较强的成分为表儿茶素IC₅₀为(0.93±0.15)μmol·L^-1、木犀草素IC₅₀为(1.96±0.19)μmol·L^-1及槲皮素IC₅₀为(2.09±0.28)μmol·L^-1,ADMET计算发现木犀草素、表儿茶素、槲皮素成药性较好。结论 采用药效团模型、分子对接、结合自由能计算及体外酶活性抑制实验,挖掘活络丸中对COX-2有抑制作用的成分,为骨关节炎中药来源的单体成分的现代化研究提供线索。     

光比浊法和血小板功能分析法评价P2Y12受体拮抗剂血小板反应性的比较研究

目的 评价光比浊法(light transmittance aggregometry, LTA)和血小板功能分析法(VerifyNow)对抗血小板药物药效学指标-血小板反应性检测结果的一致性。方法 纳入北京大学第一医院收治的,规律服用P2Y12受体抑制剂氯吡格雷或替格瑞洛的冠心病患者,在服药前采集谷浓度血样,使用LTA和VerifyNow两种检测方法对药效学指标血小板反应性进行检测,对两种检测方法结果之间的相关性进行spearman相关性分析;然后分别按LTA和VerifyNow将患者判定为血小板高反应性(high platelet reactivity, HPR)和血小板低反应性(low platelet reactivity,LPR),比较两种方法结果的一致性;最后根据抗血小板治疗药物将患者分为氯吡格雷组和替格瑞洛组,分析在不同药物组LTA和VerifyNow两种检测结果之间的相关性。结果 共纳入116名患者,其中氯吡格雷组39人,替格瑞洛组77人。血小板反应性检测结果显示LTA和VerifyNow检测结果显著相关性,r=0.565,P=3.99×10^-11。但在HPR和LPR的判定方面,两种方法一致性中等(kappa=0.403)。氯吡格雷组的两种检测方法结果具有相关性,r=0.526,P=5.89×10^-4。替格瑞洛组血小板反应性显著低于氯吡格雷组,药效更好,但两种检测方法结果不具有相关性,r=0.120,P=0.299。结论 LTA和VerifyNow检测值整体具有相关性,但在血小板反应性较低的替格瑞洛组无显著相关,两种检测方法判定出的HPR和LPR一致性中等,需要进一步改进判定的阈值。     

2种他汀类药物代谢性相互作用处方分析

HMG—CoA还原酶抑制剂（也称他汀类药物）为目前临床上常用的一类重要调血脂药。其代谢若被抑制．则引起肌病的风险大大增加。因此，了解他汀类药物的代谢特性，合理选用同类药品，有助于避免严重不良事件的发生。洛伐他汀、辛伐他汀、阿托伐他汀主要经CYP3A4代谢。普伐他汀水溶性较大，无需经CYP450代谢。氟伐他汀主要经CYP2C9代谢清除。因此，须本类药物与CYP3A4抑制剂或底物合用时应首选氟伐他汀和普伐他汀，否则有可能增加横纹肌溶解、肝功能异常或肾病等不良反应的发生。     

注射用磺苄西林钠的杂质谱研究

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

UPLC-MS/MS测定人血浆中吗啡、羟考酮、哌替啶和芬太尼的浓度研究

目的 建立同时测定人血浆中吗啡、羟考酮、哌替啶及芬太尼4种治疗药物浓度的方法,并用于治疗药物的监测。方法 血浆样本经乙腈沉淀蛋白后,以氘代吗啡作为内标,应用超高效液相色谱-串联质谱法(UPLC-MS/MS)测定药物浓度。以Agilent Eclipse XDB-C18柱(2.1 mm ×50.0 mm, 1.7 μm)为色谱柱,甲醇-水(2.0 mmol·L^-1醋酸铵)为流动相等度洗脱,流速为0.3 mL·min^-1,电喷雾离子源,应用多重反应监测模式进行正离子扫描分析。结果 吗啡、羟考酮、哌替啶及芬太尼的血药浓度在50.0～10 000.0 ng·mL^-1内线性关系良好,日内及日间精密度均小于15.0%,提取回收率及基质效应均在85.0%～115.0%内,稳定性等符合要求。结论 该方法灵敏、快速、专属性强,可以用于吗啡、羟考酮、哌替啶及芬太尼治疗药物浓度监测及药动学等方面的研究。     

(E)-N′-芳基亚甲基-4-(4-苯基嘧啶-2-基氨基)苯甲酰肼衍生物作为CDK9抑制剂的设计合成及抗HIV-1活性研究

目的 设计合成(E)-N′-芳基亚甲基-4-(4-苯基嘧啶-2-基氨基)苯甲酰肼衍生物,并对其抗HIV-1的活性进行研究。方法 4-氨基苯甲酸乙酯为起始原料,通过5步反应合成了目标化合物,采用荧光素酶(luciferase)报告基因检测了合成化合物对于HIV-1转录抑制活性。结果 目标化合物对于HIV-1的转录具有一定的抑制活性。其中化合物7p活性最优,在2 μmol·L^-1浓度下HIV-1转录抑制率为(73±0.05)%,在20 μmol·L^-1浓度下HIV-1转录活性为(90±0.01)%。进一步研究表明,化合物7p以浓度依赖性在NH1和NH2细胞中抑制HIV-1的转录活性以及下调RNA聚合酶Ⅱ CTD二号位丝氨酸磷酸化。最后,分子对接表明化合物7p与CDK9有很强的结合作用。结论 该系列化合物具有较好的抗HIV-1的活性,具有进一步研究的意义。     

延边产朝药材肾炎草的高效液相色谱指纹图谱研究

目的 建立朝药材肾炎草的HPLC-UV指纹图谱。方法 采用SunFire^TM C₁₈ (4.6 mm×250 mm, 5 μm)色谱柱;以乙腈(B)-0.4%冰醋酸溶液(A)梯度洗脱,检测波长253 nm,柱温40 ℃,进样量10 μL,流速1.0 mL · min^-1,采用对照品指认进行成分分析。结果 建立了朝药材肾炎草的HPLC-UV特征指纹图谱共有模式,标定了21个共有峰,指认了其中3个黄酮碳苷成分。10批次药材的相似度结果均大于0.95,相似程度高。结论 该方法简便,精密度、重复性和稳定性很好,特征性和专属性强,可为肾炎草药材整体质量控制提供有效手段。     

HPLC-MS/MS测定大鼠血浆中刺五加、贯叶连翘提取物主要活性成分浓度及其药动学研究

目的 建立HPLC-MS/MS测定刺五加提取物、贯叶连翘提取物主要活性成分在大鼠血浆中药物浓度的方法,应用于刺五加提取物、贯叶连翘提取物及其联用时主要活性成分在大鼠体内的药动学研究,并使用DAS 3.2.8软件对主要活性成分的药动学参数进行非房室模型拟合。方法 以Welch Ultimate XB-C₁₈(2.1 mm ×100 mm,4.6 μm)为色谱柱,乙腈(含0.1%甲酸)-0.1%甲酸水溶液(含5 mmol·L^-1乙酸铵)为流动相,以300 μL·min^-1的流速进行梯度洗脱,采用电喷雾离子源进行正负离子同时扫描,多反应监测模式进行监测。结果 所建立的方法精密度、准确度良好,提取回收率基本符合生物样品分析方法要求。结论 药动学结果表明,相比于单独给药,联合用药后刺五加苷E t_max缩短,金丝桃素ρ_max升高、t_1/2延长。该药动学结果表明,刺五加提取物、贯叶连翘提取物联用时,很可能通过增加金丝桃素血药浓度、缩短刺五加苷E的达峰时间,从而在体内产生协同抗抑郁作用。     

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??OBJECTIVE To characterize the metabolism of genistin and study its enzymatic kinetics in rat liver microsome by HPLC-MS. METHODS Genistin was incubated with rat liver microsomal incubation system. HPLC-MS method was used to characterize the metabolites. A metabolite generation method was established for quantitative analysis of genistein with sulfamethoxazole as internal standard.The enzyme kinetics parameters V_max and K_m was calculated by the GraphPad Prism 5.0 software. RESULTS The metabolites in vitro incubation system was identified as genistein.The optimal time in rat liver microsomes incubation time of 40 min,the optimal protein concentration of 1 mg??mL^-1,a substrate concentration of 50 ??mol??L^-1.The enzyme kinetics parameters of genistein were as follows: V_max=(0.104 2??0.003 3) ??mol??min^-1??mg(pro)^-1, K_m=(28.96??2.80) ??mol??L^-1. CONCLUSION The results indicate that genistin can be metabolited as the form of hydroxylation in rat liver microsome. Metabolite generation method is a reliable and simple method for determination of kinetic parameters of hepatic microsomal enzymes, and enzyme kinetic parameters obtained of genistin provide important parameters for further study.
     

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??OBJECTIVE To study the enzymatic kinetics of TM-2 in rat, Beagle dog and human liver microsomes by LC-MS/MS. METHODS TM-2 was incubated with liver microsomal incubation system. LC-MS/MS method was established for quantitative analysis of TM-2 with cabazitaxel as internal standard. The enzyme kinetics parameters V_max and K_m was calculated by the GraphPad Prism 5.0 software. RESULTS A rapid and sensitive LC-MS/MS method was developed to study the enzyme kinetics of TM-2 in rat, Beagle dog and human liver microsome. The corresponding enzymatic kinetic parameters in rat, Beagle dog and human were as follows: V_maxvalues were 16.3, 354.6 and 154.8 nmol??min^-1??mg(protein)^-1, respectively; K_m values were 25.7, 313.8 and 89.4 ??mol??L^-1, respectively; CL_int values were 0.63, 1.13 and 1.73 mL??min^-1??mg(protein)^-1, respectively. CONCLUSION The result indicates that there are species differences in the activity of metabolic enzyme and the affinity of TM-2 to the metabolic enzyme. Enzyme kinetic parameters obtained of TM-2 provide important parameters for the further study.     

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??OBJECTIVE To establish a robust, fast and convenient method for in vitro assay of rat liver CYP1A2 and CYP2D1, and explore their kinetic features.METHODS Two selective substrates including phenacetin and dextromethorphan, which are probes of CYP1A2 and CYP2D1, were chosen for liver microsomes incubation, respectively; the corresponding ultra performance liquid chromatography tandem mass spectrometry(UPLC-MS) methods were developed for kinetic studies.RESULTS The fast and convenient UPLC-MS methods with high resolution and short running time(4~5 min) were established and validated for two assays of CYP1A2 and CYP2D1 activities;both methods showed good accuracy and precision, and the values of LOQ for CYP1A2 and CYP2D1 assays could reach 0.267 and 0.007 ??mol??L^-1, respectively. The kinetic studies showed that the Michaelis constant(K_m) for CYP1A2 and CYP2D1 were (28.4??2.7) and (13.9??1.3) ??mol??L^-1, respectively. Their activities were determined to be (1.47??0.12) and (3.98??0.09) nmol??mg^-1, respectively,when the substrate concentration was 10 ??mol??L^-1.CONCLUSION UPLC Tandem MS technique is proved to be a rapid, convenient and efficient approach with high sensitivity and selectivity for the assays of CYP1A2 and CYP2D1 in drug metabolism.     

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??OBJECTIVE To investigate the risk of magnolol interfering into propofol glucuronidation in human.METHODS This study was performed in pooled microsomes from human liver, intestine and kidney (HLM, HIM, HKM, respectively). Kinetic analyses were conducted to gain the inhibition potentials of magnolol against propofol glucuronidation in HLM, HIM, and HKM.RESULTS Magnolol can potently inhibit propofol glucuronidation in HLM and HKM, following mixed inhibition kinetics with K_i values of 0.1 and 0.2 ??mol??L^-1, respectively. Different from HLM and HKM, propofol glucuronidation in HIM was not affected by the presence magnolol. CONCLUSION Magnolol is hardly to depress systemic propofol glucuronidation due to lack of inhibition of the intestinal metabolic pathway.     

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??OBJECTIVE To determin the in vitro metabolic stability of ST09 and ST10 in human liver microsomes (HLMs), and to evaluate their potential inhibitions on five HLM cytochrome P450 isoforms.METHODS A liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to assess remaining concentration of ST09 and ST10 at designed time points during the HLM incubation. Six major metabolites of cytochrome P450 were simultaneously measured with LC-MS/MS, and the inhibitory effects of ST09 and ST10 were respectively evaluated with IC₅₀ values.RESULTS ST09 was extremely unstable in vitro, and t_1/2 was less than 1 min. However, ST10, the major metabolite of ST09, was NADPH-independent metabolized in HLMs, while its t_1/2 and microsomal intrinsic clearance (CL_int) were 32 min and 0.043 mL??min^-1??mg(protein)^-1, respectively. IC₅₀ values of ST09 and ST10 on CYP3A4 (midazolam as substrate), CYP3A4 (testosterone as substrate), CYP1A2, CYP2C9, CYP2C19 and CYP2D6 were 0.42/0.25, 1.27/0.81, 24.92/18.21, 36.53/54.34, 67.64/144.90, 6.43/5.30 ??mol??L^-1, respectively.CONCLUSION ST09 and ST10 are extensively metabolized in vitro and both compounds had significant inhibition on CYP3A4 and CYP2D6.     

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??OBJECTIVE To investigate the influence of tripterygium glucoside tablet on the pharmacokinetics of atorvastatin in rats. METHODS Twelve rats were equally randomized to two groups (six rats in each group), including the atorvastatin-only group (A) and the tripterygium glucoside tablet and atorvastatin group (B). Animals in group A were administered according the oral dose of 2 mg??kg^-1; and animals in group B were administered at an oral dose of atorvastatin (2 mg??kg^-1)and tripterygium glucoside tablet (2 mg??kg^-1). Blood samples were collected into a heparinized tube via the oculi chorioideae vein at different time points after drug administration, and the plasma concentration of atorvastatin were determined using HPLC-UV. Finally, the pharmacokinetic profiles of atorvastatin were calculated and compared. RESULTS Compared with the atorvastatin-only group(A), the pharmacokinetic parameters of the tripterygium glucoside tablet and atorvastatin group(B) have changed greatly. ??_max of atorvastatin increased from (4.77??0.64) to (7.79??0.61) mg??L^-1, and AUC_0-t increased from (12.82?? 3.50) to (27.39??5.76) mg??h??L^-1, at the same time, t_max was extended from (0.25??0.03) to (0.52??0.07) h, t_1/2 was prolonged from (2.39??0.19) to (5.09??1.35) h, MRT was extended from (2.93??0.23) to (4.36??0.44)h. It indicates that the metabolism of atorvastatin may be suppressed. CONCLUSION The RESULTS indicate that tripterygium glucoside tablet could influence the pharmacokinetics of atorvastatin when atorvastatin and tripterygium glucoside tablet are used concomitantly. This study could be used for clinical medication guidance of tripterygium glucoside tablet and atorvastatin to avoid the occurrence of adverse reactions.     

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??OBJECTIVE To investigate the hepatic toxicity of 8 monomers in Polygonum multiflorum using a combination of UDP-glucuronic acid transferase 1A1(UGT1A1 enzyme). METHODS Bilirubin was used as the substrate for UGT1A1. Incubation method in RLM in vitro was adopted to test the apparent inhibition constants(K_i) of different components. Furthermore the structure-activity relationship between the 8 components and UGT1A1 was analyzed. RESULTS The inhibition effects on UGT1A1 enzyme of the 8 components were in the following sequence: emodin-8-O-glc??emodin??citreorosein??(+)-catechin??gallic acid??physcion??rhein??emodin-6-O-glc. Moreover, there was a structure-activity relationship, and it was presumed that the 6-position hydroxyl group is an active and necessary group. CONCLUSION The established method in vitro is stable and feasible. Experimental results shows that the enzyme inhibition has structural selectivity, which provides an experimental basis for predicting the enzyme inhibition activity of the analogues of components of Polygonum multiflorum.