白藜芦醇在人肝微粒体中代谢的酶动力学

目的研究白藜芦醇在体外人肝微粒体酶中的代谢以及葡糖醛酸转移酶(UGT)抑制剂对其代谢的影响。方法采用高效液相-质谱联用方法测定白藜芦醇在体外代谢系统中的代谢产物,并用Origin7.5软件分析数据,得到白藜芦醇的酶促反应动力学参数最大反应速率(Vm),米氏常数(Km),并计算UGT对白藜芦醇的内源性清除率(CLint)。通过抑制试验,观察不同浓度抑制剂对不同浓度白藜芦醇Ⅱ相代谢的影响。结果在体外代谢系统中,白藜芦醇生成两种单葡糖醛酸代谢产物M-1,M-2,初步推断其为白藜芦醇-4’,和3-葡萄糖醛酸化物。在肝微粒体中生成两种代谢产物的酶促反应的Vm和Km分别为M-1:(1.08±0.06)nmol.min-1.mg-1和(192.05±30.46)μmol.L-1;M-2:(2.20±0.10)nmol.min-1.mg-1和(34.82±6.95)μmol.L-1,白藜芦醇代谢为M-2的代谢速率明显高于M-1。UGT抑制剂姜黄素和槲皮素都能显著抑制白藜芦醇的代谢。结论代谢产物M-2的生成是白藜芦醇在肝微粒体中的主要消除途径。姜黄素、槲皮素在体外对白藜芦醇的Ⅱ相代谢有显著的抑制作用,对临床研究药物的相互作用有很大指导意义。     

阿卡宁的氧葡萄糖醛酸化代谢通路研究

目的对紫草素的立体异构单体阿卡宁的氧葡萄糖醛酸代谢通路进行研究和表征。方法采用液相色谱和质谱联用方法检测阿卡宁和其葡萄糖醛酸化代谢产物;将阿卡宁在人肝微粒体(HLM)、人肾微粒体(HKM)以及重组人类葡萄糖醛酸转移酶(UGT)中孵育,观察代谢轮廓、筛选参与催化的重组单酶、考察酶动力学;通过相关性分析以及化学抑制实验阐明UGT单酶对阿卡宁的选择性。结果阿卡宁在含尿苷5’-二磷酸葡萄糖醛酸(UDPGA)的HLM孵育中,可以检测到1个UGT代谢产物。UGT单酶筛选发现UGT1A9高选择性催化阿卡宁。酶动力学研究显示在HLM、HKM和UGT1A9中阿卡宁的UGT代谢都呈底物抑制模式,并且表观亲和常数(Km)在3.75~4.50μmol/L。阿卡宁和已知UGT1A9探针底物异丙酚在12例个体人肝中的UGT代谢具有很好的相关性,R2为0.88。化学抑制实验显示在HLM中,厚朴酚和尼氟灭酸对阿卡宁的UGT代谢具有明显的抑制作用;睾酮、雷公藤红素和尼罗替尼对阿卡宁的UGT代谢均无明显抑制作用。结论 UGT代谢是阿卡宁(紫草素)在人体的重要代谢途径之一,阿卡宁是人类UGT1A9的一个高选择性探针底物。     

补骨脂定的肝肠微粒体代谢动力学、代谢酶表型及种属差异研究

研究补骨脂定在人肝微粒体(HLM)和肠微粒体(HIM)的代谢活性,明确参与补骨脂定代谢的细胞色素P450酶(CYPs)和尿苷二磷酸葡萄糖醛酸转移酶(UGTs)及补骨脂定体外代谢的种属差异。将不同浓度的补骨脂定溶液分别与HLM和HIM共同孵育,经HLM孵育可以产生2个氧化产物(M1和M2)和2个葡萄糖醛酸结合物(G1和G2),在HIM中仅产生M1和G1;在HLM和HIM中,补骨脂定代谢生成M1的固有清除率(CLint)分别为104.3、57.6μL·min~(-1)·mg~(-1),生成G1的CLint分别为543.3、75.9μL·min~(-1)·mg~(-1)。利用12种CYPs和12种UGTs酶,分别与不同浓度的补骨脂定溶液共同孵育,结果显示,CYP1A1(39.5μL·min~(-1)·mg~(-1))、CYP2C8(88.0μL·min~(-1)·mg~(-1))、CYP2C19(166.7μL·min~(-1)·mg~(-1))、CYP2D6(9.1μL·min~(-1)·mg~(-1))是生成M1的关键CYPs代谢酶,而CYP2C19(42.0μL·min~(-1)·mg~(-1))是生成M2的重要亚型酶; UGT1A1(1 184.4μL·min~(-1)·mg~(-1))、UGT1A7(922.8μL·min~(-1)·mg~(-1))、UGT1A8(133.0μL·min~(-1)·mg~(-1))、UGT1A9(348.6μL·min~(-1)·mg~(-1))、UGT2B7(118.7μL·min~(-1)·mg~(-1))重点参与G1的生成,而UGT1A9(111.3μL·min~(-1)·mg~(-1))是G2生成的关键亚型酶。采用猴肝微粒体(MkLM)、大鼠肝微粒体(RLM)、小鼠肝微粒体(MLM)、狗肝微粒体(DLM)和猪肝微粒体(MpLM),分别与不同浓度的补骨脂定溶液共同孵育,结果显示,补骨脂定的Ⅰ相代谢和葡萄糖醛酸化代谢均表现出显著的种属差异。总体来说,补骨脂定在肝肠微粒体均可以发生较强的代谢; CYP1A1、CYP2C8、CYP2C19、CYP2D6与UGT1A1、UGT1A7、UGT1A8、UGT1A9、UGT2B7是参与其代谢的关键亚型酶;大鼠和猪分别是研究补骨脂定Ⅰ相代谢和葡萄糖醛酸化代谢合适的模式动物。     

乙烯苷体外肝微粒体中潜在毒性作用

目的：以胆红素代谢过程中UDP-葡萄糖醛酸转移酶1A1（UGT1A1酶）介导的胆红素葡萄糖醛酸结合环节为切入点,考察何首乌中含量高的二苯乙烯苷类（顺式、反式二苯乙烯苷）潜在肝毒性,探索何首乌致肝毒性物质基础。方法：以胆红素为UGT1A1酶底物,以表观抑制常数Ki为评价指标,采用体外肝微粒体孵育法,启动Ⅱ相代谢反应,考察顺式、反式二苯乙烯苷原型成分的抑制作用;启动Ⅰ相代谢反应,考察代谢产物及原型成分的抑制作用,推测待测物的潜在毒性作用。结果：当仅启动Ⅱ相反应时,顺式、反式二苯乙烯苷均以原型形式直接作用于UGT1A1酶,两个单体分别表现出中等抑制和弱抑制作用,抑制类型均为竞争型抑制;当同时启动Ⅰ、Ⅱ两相反应时,两个待测单体对UGT1A1酶的抑制作用均消失,提示两个单体存在Ⅰ相代谢过程,并且其Ⅰ相代谢产物对UGT1A1酶无抑制作用。结论：本实验初步证明何首乌中顺式、反式二苯乙烯苷原型成分对UGT1A1存在抑制作用,经由Ⅰ相代谢后,对Ⅱ相代谢酶UGT1A1抑制作用消失,肝毒性风险降低。     

基于UGT1A1酶抑制探讨何首乌中顺(反)式二苯乙烯苷体外肝微粒体中潜在毒性作用

目的:以胆红素代谢过程中UDP-葡萄糖醛酸转移酶1A1(UGT1A1酶)介导的胆红素葡萄糖醛酸结合环节为切入点,考察何首乌中含量高的二苯乙烯苷类(顺式、反式二苯乙烯苷)潜在肝毒性,探索何首乌致肝毒性物质基础。方法:以胆红素为UGT1A1酶底物,以表观抑制常数Ki为评价指标,采用体外肝微粒体孵育法,启动Ⅱ相代谢反应,考察顺式、反式二苯乙烯苷原型成分的抑制作用;启动Ⅰ相代谢反应,考察代谢产物及原型成分的抑制作用,推测待测物的潜在毒性作用。结果:当仅启动Ⅱ相反应时,顺式、反式二苯乙烯苷均以原型形式直接作用于UGT1A1酶,两个单体分别表现出中等抑制和弱抑制作用,抑制类型均为竞争型抑制;当同时启动Ⅰ、Ⅱ两相反应时,两个待测单体对UGT1A1酶的抑制作用均消失,提示两个单体存在Ⅰ相代谢过程,并且其Ⅰ相代谢产物对UGT1A1酶无抑制作用。结论:本实验初步证明何首乌中顺式、反式二苯乙烯苷原型成分对UGT1A1存在抑制作用,经由Ⅰ相代谢后,对Ⅱ相代谢酶UGT1A1抑制作用消失,肝毒性风险降低。     

基于体外肝代谢考察大黄素甲醚肝毒性作用

以胆红素代谢过程中UDP-葡萄糖醛酸转移酶1A1(UGT1A1酶)介导的胆红素葡萄糖醛酸结合环节为切入点,评价大黄素甲醚潜在肝毒性风险。实验以胆红素为UGT1A1酶底物,以表观抑制常数K_i为评价指标,采用体外肝微粒体孵育法,启动Ⅱ相代谢反应,考察大黄素甲醚原型成分的抑制作用,启动Ⅰ,Ⅱ相代谢反应,考察代谢产物及原型成分的综合抑制作用。结果显示仅启动Ⅱ相反应时,大黄素甲醚以原型形式直接作用于UGT1A1酶,表现为弱抑制,抑制类型为混合型抑制;同时启动Ⅰ,Ⅱ相反应时,大黄素甲醚对UGT1A1酶的抑制作用变为强抑制,抑制类型为混合型抑制,提示大黄素甲醚存在Ⅰ,Ⅱ相代谢过程,并且其代谢产物大黄素葡萄糖苷结合物及大黄素葡萄糖醛酸化物对UGT1A1酶抑制作用较强。该研究所得结果初步证明大黄素甲醚原型对UGT1A1无明显抑制作用,经由Ⅰ,Ⅱ相代谢后抑制作用增强,推测其代谢产物为引发肝毒性的主要成分。     

人尿苷二磷酸葡糖醛酸转移酶参与马钱子碱和士的宁体外代谢研究北大核心CSCD

目的考察参与士的宁和马钱子碱Ⅱ相代谢的主要人尿苷二磷酸葡糖醛酸转移酶(UGTs)亚型,为预测其与其他药物代谢性相互作用提供理论借鉴。方法士的宁和马钱子碱分别与大鼠肝微粒体(RLMs)、人肝微粒体(HLMs)和12种主要人重组UGTs亚酶进行孵育,采用高效液相色谱-串联质谱(HPLC-MS/MS)法检测士的宁和马钱子碱葡糖醛酸代谢产物,考察参与其体外Ⅱ相代谢UGTs亚酶类型。结果马钱子碱和士的宁均在HLMs里生成1个单葡糖醛酸代谢产物,而在RLMs中未产生,单酶试验中二者均只在UGT1A4重组酶中发生代谢。结论马钱子碱和士的宁在人鼠代谢中存在种属差异,UGT1A4为其主要代谢亚酶。该研究结果可为临床预防马钱子因药物代谢性相互作用引发的不良反应提供理论和实验依据。     

人尿苷二磷酸葡糖醛酸转移酶参与马钱子碱和士的宁体外代谢研究

目的考察参与士的宁和马钱子碱Ⅱ相代谢的主要人尿苷二磷酸葡糖醛酸转移酶(UGTs)亚型,为预测其与其他药物代谢性相互作用提供理论借鉴。方法士的宁和马钱子碱分别与大鼠肝微粒体(RLMs)、人肝微粒体(HLMs)和12种主要人重组UGTs亚酶进行孵育,采用高效液相色谱-串联质谱(HPLC-MS/MS)法检测士的宁和马钱子碱葡糖醛酸代谢产物,考察参与其体外Ⅱ相代谢UGTs亚酶类型。结果马钱子碱和士的宁均在HLMs里生成1个单葡糖醛酸代谢产物,而在RLMs中未产生,单酶试验中二者均只在UGT1A4重组酶中发生代谢。结论马钱子碱和士的宁在人鼠代谢中存在种属差异,UGT1A4为其主要代谢亚酶。该研究结果可为临床预防马钱子因药物代谢性相互作用引发的不良反应提供理论和实验依据。     

淫羊藿次苷Ⅱ葡萄糖醛酸代谢产物的制备与表征

目的:鉴定淫羊藿次苷Ⅱ葡萄糖醛酸代谢产物,并进行制备与结构表征。方法:应用人肝微粒体孵育体系对淫羊藿次苷Ⅱ葡糖醛酸结合代谢途径进行鉴定,并应用重组UGT1A1高效制备葡萄糖醛酸代谢产物,最后经NMR对该产物结构进行表征。结果:淫羊藿次苷Ⅱ在人肝微粒体中的主要代谢产物为淫羊藿次苷Ⅱ-7-O-葡萄糖醛酸代谢产物。结论:采用体外孵育方法可以高效特异地制备淫羊藿次苷Ⅱ的葡萄糖醛酸代谢产物。     

白杨黄素和天然香豆素类对大鼠和人原代培养的肝细胞UGT1A1和UGT1A6活性的影响

二磷酸尿核苷葡萄糖醛酸基转移酶(UGTs)是许多组织细胞内质网中的一种二相药物代谢酶,其家族中的UGT1和UGT2亚型在有毒物质的代谢过程中具有重要的生理意义。研究了白杨黄素和6种天然香豆素类化合物(缴形酮、香豆素、勒素、cis- avicennol、香柠檬烯和前胡醚)对人和大鼠原代培养的肝细胞中UGT1A1和(或)UGT1A6的影响。     

Identification and interspecies characterization of UDP-glucuronosyltransferase isoforms catalyzing acacetin glucuronidation using recombinant UGT enzymes and microsomes

ObjectiveTo explore the glucuronic acid metabolism of acacetin in human liver and intestinal microsomes to better characterize human uridine 5′-diphospho (UDP)-glucuronosyltransferase (UGT) isoforms. In addition, interspecies comparisons were performed to identify the most appropriate experimental animal model for an in vivo study.MethodsLiquid chromatography tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) were used to confirm the successful biosynthesis of acacetin-7-O-glucuronide. Human isoforms of UGT and isozyme-specific chemical inhibitors were used for recombinant assays. Acacetin glucuronidation kinetics were assessed by combining acacetin with recombinant human UGT isoforms or with microsomes from humans or experimental animals. Kinetic differences between species were assessed in vitro using the same approach.ResultsWe identified multiple UGT isoforms that facilitated acacetin glucuronidation, and found that UGT1A1 was the major isoform that catalyzed this process. Acacetin-7-O-glucuronide formation exhibited clear substrate inhibition kinetics when combined with recombinant UGTs or with liver/intestinal microsomes derived from humans, monkeys, rats, mice, dogs, or pigs. Intrinsic metabolic clearance values of human intestinal microsomes were two-fold greater than those of human liver microsomes. Among the evaluated species, the K_m value of dog microsomes (0.86 μM) was greatest in acacetin glucuronidation, while mice exhibited the highest CL_int value, 5.05 mL/min/mg. The CL_int values of microsomes derived from monkeys and minipigs were 1.99 mL/min/mg and 2.12 mL/min/mg, respectively, exhibiting similar intrinsic metabolic clearance activity to that observed in humans.ConclusionMonkey may represent a suitable model for experimental studies of acacetin pharmacokinetics owing to a high sequence homology of UGT1A1 and similar UGT1A1 glucuronidation activity to humans.     

Comparison of Inhibition Capability of Scutellarein and Scutellarin Towards Important Liver UDP‐Glucuronosyltransferase (UGT) Isoforms

Scutellarin is an important bioactive flavonoid extracted from Erigeron breviscapus (Vant.) Hand‐Mazz, and scutellarein is the corresponding aglycone of scutellarin. The present study aims to compare the inhibition potential of scutellarin and scutellarein towards several important UDP‐glucuronosyltransferase (UGT) isoforms, including UGT1A1, UGT1A6, UGT1A9 and UGT2B7. It was demonstrated that scutellarein exerted stronger inhibition towards the tested UGT isoforms than scutellarin. Furthermore, the inhibition kinetic type and parameters (K_i) were determined for the scutellarein's inhibition towards these UGT isoforms. Competitive inhibition of scutellarein towards all these UGT isoforms was demonstrated, and the K_i values were calculated to be 0.02, 5.0, 5.8 and 35.9 μM for UGT1A1, 1A6, 1A9 and 2B7, respectively. Using in vivo maximum plasma concentration of scutellarein in rat, the in vitro–in vivo extrapolation was performed to predict in vivo situation, indicating the most possible in vivo adverse effects due to the inhibition of scutellarein towards UGT1A1. All these results remind us to monitor the utilization of scutellarin and scutellarein, and the herbs containing these two components. Copyright © 2013 John Wiley & Sons, Ltd.     

蛋白激酶抑制剂调控木犀草素与葡萄糖醛酸结合的研究

目的探究蛋白激酶抑制剂对木犀草素与葡萄糖醛酸结合的影响。方法分别将不同浓度的蛋白激酶抑制剂(姜黄素和钙感光蛋白C)与LS174T细胞共孵育1 h后裂解细胞,获得的细胞S9成分与木犀草素进行体外孵育,用高效液相色谱法测定孵育液中葡萄糖醛酸结合产物。通过Real-time PCR和Western blot分别测定处理后的LS174T细胞中UGT1A在mRNA水平和蛋白水平的表达变化。结果木犀草素与经蛋白激酶抑制剂处理后的细胞共孵育,其葡萄糖醛酸结合受到抑制。而Real-time PCR和Western blot均表明,蛋白激酶抑制剂对UGT1A的表达无影响。结论蛋白激酶抑制剂可抑制木犀草素与葡萄糖醛酸的结合,而这种抑制作用是间接的。蛋白激酶抑制剂很可能通过调控UGTs磷酸化修饰而改变UGTs活性进而导致木犀草素与葡萄糖醛酸结合减少。     

Deglycosylation of Liquiritin Strongly Enhances its Inhibitory Potential Towards UDP‐Glucuronosyltransferase (UGT) Isoforms

The detailed mechanisms on licorice–drug interaction remain to be unclear. The aim of the present study is to investigate the inhibition of important UGT isoforms by two important ingredients of licorice, liquiritin, and liquiritigenin. The results showed that liquiritigenin exhibited stronger inhibition towards all the tested UGT isoforms than liquiritin. Data fitting using Dixon and Lineweaver–Burk plots demonstrated the competitive inhibition of liquiritigenin towards UGT1A1 and UGT1A9‐mediated 4‐MU glucuronidation reaction. The inhibition kinetic parameters (K_i) were calculated to be 9.1 and 3.2 μM for UGT1A1 and UGT1A9, respectively. Substrate‐dependent inhibition behaviour was also observed for UGT1A1 in the present study. All these results will be helpful for understanding the deep mechanism of licorice–drug interaction. However, when translating these in vitro parameters into in vivo situations, more complex factors should be considered, such as substrate‐dependent inhibition of UGT isoforms, the contribution of UGT1A1 and UGT1A9 towards the metabolism of drugs, and many factors affecting the abundance of ingredients in the licorice. Copyright © 2012 John Wiley & Sons, Ltd.     

The Inhibition of UDP‐Glucuronosyltransferase (UGT) Isoforms by Praeruptorin A and B

Praeruptorin A (PA) and B (PB) are two important compounds isolated from Bai‐hua Qian‐hu and have been reported to exert multiple biochemical and pharmacological activities. The present study aims to determine the inhibition of PA and PB on the activity of important phase II drug‐metabolizing enzymes uridine 5'‐diphospho‐glucuronosyltransferase (UGTs) isoforms. In vitro UGT incubation system was used to determine the inhibition potential of PA and PB on the activity of various UGT isoforms. In silico docking was performed to explain the inhibition difference between PA and PB towards the activity of UGT1A6. Inhibition behaviour was determined, and in vitro–in vivo extrapolation was performed by using the combination of in vitro inhibition kinetic parameter (K_i) and in vivo exposure level of PA. Praeruptorin A (100 μM) exhibited the strongest inhibition on the activity of UGT1A6 and UGT2B7, with 97.8% and 90.1% activity inhibited by 100 μM of PA, respectively. In silico docking study indicates the significant contribution of hydrogen bond interaction towards the stronger inhibition of PA than PB towards UGT1A6. Praeruptorin A noncompetitively inhibited the activity of UGT1A6 and competitively inhibited the activity of UGT2B7. The inhibition kinetic parameter (K_i) of PA towards UGT1A6 and UGT2B7 was calculated to be 1.2 and 3.3 μM, respectively. The [I]/K_i value was calculated to be 15.8 and 5.8 for the inhibition of PA on UGT1A6 and UGT2B7, indicating high inhibition potential of PA towards these two UGT isoforms in vivo. Therefore, closely monitoring the interaction between PA and drugs mainly undergoing UGT1A6 or UGT2B7‐catalyzed metabolism is very necessary. Copyright © 2016 John Wiley & Sons, Ltd.     

The Inhibitory Effect of 20(S)‐Protopanaxatriol (ppt) Towards UGT1A1 and UGT2B7

Ginseng, a commonly used natural product, has been frequently reported to induce herb–drug interaction with many clinical drugs. The intestinal bacterial metabolites of ginsenosides have been widely regarded as the substance basis for ginseng–drug interactions. To date, little is known about the inhibitory effect of intestinal bacterial metabolites of ginsenosides towards UDP‐glucuronosyltransferases (UGTs). In vitro investigation of the inhibition of 20(S)‐protopanaxatriol (ppt) towards UGT1A1 and UGT2B7 was carried out. The results showed that ppt exhibited strong noncompetitive inhibition towards UGT1A1 and competitive inhibition towards UGT2B7. The inhibition kinetic parameters (K_i) were calculated to be 8.8 and 2.2 μM for UGT1A1 and UGT2B7, respectively. Using the maximum plasma concentration of ppt, the alteration of area under the concentration–time curve was calculated to be 20% and 70% respectively for UGT1A1‐mediated and UGT2B7‐mediated metabolism. However, given that the varied contribution of these two UGT isoforms towards drug metabolism and the influence of herb complexity and individual difference, the explanation of these results should be paid more caution. Copyright © 2012 John Wiley & Sons, Ltd.     

In Vitro Characterization of Glucuronidation of Vanillin: Identification of Human UDP‐Glucuronosyltransferases and Species Differences

Vanillin is a food flavoring agent widely utilized in foods, beverages, drugs, and perfumes and has been demonstrated to exhibit multiple pharmacological activities. Given the importance of glucuronidation in the metabolism of vanillin, the UDP‐glucuronosyltransferase conjugation pathway of vanillin was investigated in this study. Vanillin glucuronide was identified by high‐performance liquid chromatography‐tandem mass spectrometry (HPLC‐MS/MS) and a hydrolysis reaction catalyzed by β‐glucuronidase. The kinetic study showed that vanillin glucuronidation by HLMs and HIMs followed Michaelis‐Menten kinetics and the kinetic parameters were as follows: 134.9 ± 13.5 μM and 81.3 ± 11.3 μM for K_m of HLMs and HIMs, 63.8 ± 2.0 nmol/min/mg pro and 13.4 ±2.0 nmol/min/mg pro for V_max of HLMs and HIMs. All UDP‐glucuronosyltransferase (UGT) isoforms except UGT1A4, 1A9, and 2B7 showed the capability to glucuronidate vanillin, and UGT1A6 exerted the higher V_max/K_m values than other UGT isoforms for the glucuronidation of vanillin when assuming expression of isoforms is similar in recombinant UGTs. Kinetic analysis using liver microsomes from six studied speices indicated that vanillin had highest affinity for the monkey liver microsomes enzyme (K_m = 25.6 ± 3.2 μM) and the lowest affinity for the mice liver microsomes enzyme (K_m = 149.1 ± 18.4 μM), and intrinsic clearance was in the following order: monkey > dog > minipig > mice > rat ~ human. These data collectively provided important information for understanding glucuronidation of vanillin. Copyright © 2012 John Wiley & Sons, Ltd.     

Investigation of UDP-glucuronosyltransferases (UGTs) inhibitory properties of carvacrol

UDP‐glucuronosyltransferases (UGTs), the most important phase II drug metabolizing enzymes (DMEs), could metabolize many drugs and various endogenous substances including bilirubin, steroid hormones, thyroid hormones, bile acids and fat‐soluble vitamins. Evaluation of the inhibitory effects of compounds on UGTs is clinically important because inhibition of UGT isoforms could not only result in serious drug–drug interactions (DDIs), but also induce metabolic disorders of endogenous substances. The aim of the present study was to investigate the inhibitory effects of carvacrol on major UGT isoforms. The results showed that carvacrol could inhibit the activity of UGT1A9 with negligible effects on other UGT isoforms. When 4‐methylumbelliferone (4‐MU) was used as a nonspecific probe substrate and recombinant UGT enzymes were utilized as an enzyme resource, the inhibition of UGT1A9 was best fit to the competitive type and the inhibition kinetic parameter (K_i) was calculated to be 5.7 µ m . Furthermore, another specific probe substrate, propofol, was employed to determine the inhibitory kinetics of UGT1A9, and the results demonstrated that the inhibitory type was noncompetitive. The inhibition kinetic parameter (K_i) was determined to be 25.0 µ m . Because this substrate‐dependent inhibition of UGT1A9 might confuse the in vitro–in vivo extrapolation, these in vitro inhibition kinetic parameters should be interpreted with special caution. Copyright © 2011 John Wiley & Sons, Ltd.     

LC-MS-MS分析白藜芦醇在大鼠胆汁和尿液中的代谢产物

目的： 研究白藜芦醇的体内代谢产物。 方法： 采用液质联用法,通过分析原型化合物及代谢产物的液相分析保留时间、二级质谱裂解数据以及紫外吸收光谱,推测单次给药白藜芦醇后的代谢产物。色谱条件：Zorbax SB-C₁₈色谱柱（4.6 mm×250 mm,5 μm）,Zorbax SB-C₁₈ 保护柱（4 mm×20 mm,5 μm）,流动相（A）乙腈-（B）水梯度洗脱：0～20 min, 3%～18%A,20～40 min,18%～35%A,流速1.0 mL·min^-1,检测波长320 nm,柱温30℃。质谱条件Finnigan Surveyor^TM HPLC-Finnigan TSQ,ESI源,Xcalibur 1.2数据软件 LCQ数据处理软件,ESI离子源,负离子扫描模式扫描范围m/z 100～800,鞘气流15 arb,辅助气流15 unit,喷雾电压4 000 V,毛细管温度300℃。 结果： 推测了大鼠尿液和胆汁中5个代谢产物的相对分子质量、保留时间、紫外最大吸收波长、二级质谱裂解规律及化学结构。 结论： 在尿液排泄中以硫酸酯化为主要的代谢途径,而在胆汁排泄中以葡萄糖醛酸化为主要的代谢方式。     

聚乙二醇400对黄芩苷及其主代谢物胆汁排泄的影响

目的:考察药用辅料聚乙二醇400(PEG400)对黄芩苷及其主代谢物黄芩素6-O-β-D-葡萄糖醛酸苷(B6G)胆汁排泄的影响,并分析其作用机制。方法:大鼠随机分为黄芩苷+水组与黄芩苷+PEG400组,利用10%水合氯醛(剂量4 mL·kg-1)腹腔注射诱导麻醉,制作大鼠胆管插管模型,待大鼠完全清醒后,以168 mg·kg-1剂量的黄芩苷分别给大鼠灌胃相应的黄芩苷水溶液和黄芩苷PEG400溶液,收集给药后0~12 h的胆汁,使用UPLC-MS/MS测定不同时间段药物经胆汁排泄的浓度,采用Thermo Hypersil GOLD C18色谱柱(2.1 mm×100 mm,1.9μm),流动相乙腈(A)-0.1%甲酸水溶液(B)梯度洗脱(0~9 min,90%~27%B;9~10 min,27%~90%B;10~12 min,90%B),流速0.3 mL·min^-1,柱温30℃,进样量5μL;质谱条件为电喷雾离子源(ESI),正离子检测模式。利用体外孵育法和酶联免疫吸附测定(ELISA)研究PEG400对尿苷二磷酸葡萄糖醛酸转移酶(UGT)1A8和UGT1A9活性及二者在大鼠肝脏中表达的影响。结果:与黄芩苷+水组相比,黄芩苷+PEG400组大鼠12 h内胆汁中黄芩苷及其主代谢物B6G的累积排泄量分别增加了1.8,2.1倍;PEG400分别使UGT1A8与UGT1A9的酶活性提高了2.0,1.5倍,使肝脏中UGT1A8与UGT1A9的质量分数提高了2.2,1.3倍。结论:PEG400可通过提高UGT1A8与UGT1A9的活性和表达来显著增加黄芩苷及其主代谢物B6G的胆汁排泄,且对B6G胆汁排泄的促进作用大于原形药物黄芩苷,可为PEG400的临床合理应用及黄芩苷等黄酮类药物新剂型的设计提供依据。