CN112725408B - 一种ugt酶活性检测方法及其应用 - Google Patents
一种ugt酶活性检测方法及其应用 Download PDFInfo
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Abstract
本发明提供了一种非用于疾病诊断治疗的UGT酶活性的检测方法及其在筛选和评价UGT酶调控剂中的应用。该检测方法以麦冬异黄酮A作为底物,通过液相‑紫外法或液相‑质谱法定量检测麦冬异黄酮A的转化速率或其7‑O‑葡萄糖醛酸产物的生成速率来检测UGT酶的活性。本发明所述方法具有灵敏度高、稳定性强、重复性好、简单易操作等特点;利用该方法对多种已报道的UGT酶调控剂进行了筛选与评价,有极高的准确性,具有非常良好的应用前景。
Description
技术领域
本发明属于医药技术领域,具体涉及一种非用于疾病诊断治疗的UGT酶活性的检测方法,以及该检测方法在UGT酶调控剂筛选与评价中的应用。
背景技术
尿苷二磷酸葡萄糖醛酸转移酶(UGT)超家族是机体内重要的Ⅱ相代谢酶,其通过催化化合物与辅因子尿苷二磷酸葡萄糖醛酸(UDPGA)结合,从而增加底物的亲水性,使化合物能更有效地从尿液或胆汁中排出体外,这是机体的一个重要的解毒过程。哺乳动物的UGT可分为4个家族:UGT1,UGT2,UGT3和UGT8。其中参与药物结合代谢的多为UGT1A和UGT2B亚家族的成员。
作为机体最重要的II相代谢酶,UGT酶在各种异源生物(如治疗药物及其Ⅰ相代谢产物,食品化学成分或环境毒素)的代谢清除和解毒中发挥了关键作用。除异源物代谢外,UGT酶还参与许多内源性物质(如胆红素,类固醇和胆汁酸)的葡萄糖醛酸化代谢。当UGT酶出现功能障碍或被强效抑制后则可能会引发内源物代谢障碍及药物/草药相互作用(DDI/HDI),给人类健康带来不良影响。反之,部分UGT酶(如UGT1A1)的激动剂可加速胆红素等毒物的代谢清除,进而缓解疾病的发生发展进程。因此,业界一直期望能开发出UGT酶调控剂(抑制剂或激动剂)的高效筛选与评价方法。
由于UGT酶氨基酸序列的高度一致导致其底物谱高度重叠,因此UGT酶的特异性探针底物报道极少。到目前为止,仅有少数UGT亚型酶(包括UGT1A1,UGT1A4,UGT1A9和UGT2B7)具有公认的特异性底物。而对于其他UGT亚型酶的活性检测,只能依靠传统非荧光底物4-甲基伞形酮(4-MU),其检测通量低,样本前处理及检测过程操作繁琐复杂,无法同步开展目标物对多种UGT亚型酶抑制能力的规模化筛选与评价。
因此,设计研发UGT酶活性的非疾病诊断治疗的检测方法,以及该检测方法在UGT酶调控剂筛选与评价中的应用对于UGT介导的药物相互作用评价具有重要意义。
发明内容
本发明属于医药技术领域,具体涉及一种非用于疾病诊断治疗的UGT酶活性的检测方法,以及该检测方法在UGT酶调控剂筛选与评价中的应用。该方法以麦冬高异黄酮A(MOA)为底物,在生理条件下,其可被13种人源UGT酶催化生成麦冬高异黄酮A的7-O-葡萄糖醛酸(MOAG),该产物可被液相-紫外或液相-质谱检测。借助该探针反应,以重组表达的哺乳动物UGT酶为酶源,可同步实现待测化合物对13种人源UGT酶的调控能力的筛选与评价。上述筛选与评价方法借助液相-紫外检测***(LC-UV)或液相-质谱检测***(LC-MS)开展,该方法具有检测灵敏、操作简便、检测精密度和稳定性高、抗干扰能力强及廉价高效等优点,具有良好的应用前景。
本发明提供一种非用于疾病诊断治疗的UGT酶活性的检测方法,采用麦冬异黄酮A作为底物,通过定量检测麦冬异黄酮A的转化速率或其7-O-葡萄糖醛酸产物的生成速率来检测UGT酶的活性。
本发明的方法包括如下步骤:
(1)将三羟甲基氨基甲烷-盐酸缓冲液与MgCl2、麦冬高异黄酮A、UGT酶混合,预孵育3-5分钟,孵育温度为20-60℃,反应液pH值为5-10;
(2)在步骤(1)所得的反应液中加入尿苷二磷酸葡萄糖醛酸,反应10-60分钟,得到麦冬高异黄酮A的7-O-葡萄糖醛酸产物;
(3)在步骤(2)所得的反应液中加入冰乙腈或冰甲醇,沉淀蛋白后离心,得到上清液;
(4)用液相-紫外***或液相-质谱***检测步骤(3)所得上清液中麦冬高异黄酮A的转化速率或麦冬高异黄酮A的7-O-葡萄糖醛酸产物的生成速率。
反应方程式如式1所示:
本发明中,UGT酶选自UGT1A1,UGT1A3,UGT1A4,UGT1A6,UGT1A7,UGT1A8,UGT1A9,UGT1A10,UGT2B4,UGT2B7,UGT2B10,UGT2B15或UGT2B17中的一种或几种。
本发明中,当步骤(4)使用液相-紫外***时,其检测条件为,以十八烷基硅烷柱为固定相,以0.2%甲酸水(B)和乙腈(A)为流动相,采用梯度洗脱方式分离,紫外检测波长为300nm。
本发明中,当步骤(4)使用液相-质谱***时,在步骤(3)中需要加入内标4-甲基伞型酮-β-D-葡糖苷酸。
本发明中,当步骤(4)使用液相-质谱***时,其检测条件为,以十八烷基硅烷柱为固定相,以0.2%甲酸水(B)和乙腈(A)为流动相,流速设置为0.4ml/min,进样量为2μL,采用梯度洗脱方式分离:0-0.5min,98%B,0.5-0.6min,98%-15%B;0.6-1.6min,15%B;1.6-1.7min,98%B;1.7-3min,98%B;采用电喷雾离子源、负离子、多反应监测模式;氮气为雾化气和干燥气;高纯氮气为碰撞气,压力是0.1MPa;质谱仪器参数设置如下:离子化电压(Is):-4500V;喷撞气(CAD):Medium;气帘气(CUR):20psi,喷雾气(GS1):20psi,离子源温度(TEM):450℃,去簇电压(DP):-80V,碰撞电压(CE):15V;用于定量分析的检测离子为:MOAG质荷比517.138→341.105;内标4-甲基伞型酮-β-D-葡糖苷酸质荷比351.0→175.0。
本发明的第二目的是提供该检测方法在UGT酶调控剂筛选与评价中的应用,所述方法还包括在步骤(1)中加入待测化合物以及在步骤(4)中对待测化合物的调控作用进行评价。
本发明中,当待测化合物使UTG酶活性上升时为激动剂;当待测化合物使UTG酶活性下降时为抑制剂。
本发明具有以下优势:
(1)检测灵敏:广谱底物麦冬高异黄酮A的葡萄糖醛酸化反应可被多种UGT酶催化代谢生成单一代谢产物,且该底物与UGT酶的亲和力较好,检测方法中所使用的LC-UV或LC-MS检测器也较为灵敏,所需样品量极少,在实际操作中可以减少实验材料的用量,降低实验成本。
(2)稳定性强:本发明中,产物与底物不会相互反应,不会相互干扰,且所建立的方法使用LC-UV或LC-MS检测,能实现对样本中存在的各类物质实现良好的分离,避免了杂质对样本的干扰,在4℃条件下利用该方法进行实验样本处理与测定,至少两天不会有明显响应信号强度变化,方便实时定量监测其葡萄糖醛酸化反应过程。
(3)操作简单、重复性好:本发明利用天然产物麦冬高异黄酮A构建,原料可直接购买,且UGT酶活性的检测及UGT酶调控剂的筛选方法也具有操作便捷、耗费低等特点,经济适用性较好。且该广谱底物反应本身较为稳定,实验重复性较好。
(4)检测的UGT酶种类多:该方法能实现对人体所有13种参与药物代谢的UGT酶活性的检测,与使用4-甲基伞形酮为底物的传统方法相比,其能检测UGT1A4和UGT2B10的O-葡萄糖醛酸化活性,并在相同条件下对十三种酶的调控剂进行筛选与评价,避免了使用不同底物进行筛选评价时的实验误差。
本发明基于天然产物筛选发现的广谱底物麦冬高异黄酮A构建,该底物仅生成一个单葡萄糖醛酸化产物代谢产物单一、能检测13种UGT亚型酶的活性。本发明具有灵敏度高、稳定性强、重复性好、简单易操作等特点。对多种已报道的UGT酶选择性抑制剂进行了筛选与重评价,证明该筛选方法具有极高的准确性,具有非常良好的应用前景。
附图说明
图1.实施例1中MOA发生葡萄糖醛酸化反应的速率图。
图2.实施例2中MOAG的质谱图。
图3.实施例3中稳定性考察图
图4.实施例4中液相-紫外法检测MOA葡萄糖醛酸化反应过程图。
图5.实施例5中液相-质谱法检测MOA葡萄糖醛酸化反应过程图。
图6.实施例2中MOAG的核磁鉴定图,其中,图(a)是氢谱图,图(b)是碳谱图。
图7.实施例6中基于紫外检测***对UGT酶抑制剂进行筛选与评价的IC50图,其中--表示没有抑制或抑制很微弱,IC50>100μM。
图8.实施例7中使用LC-MS检测的质谱图。
图9.实施例8中利用LC-UV检测***及建立的方法对化合物黄豆黄素激活UGT酶效果的筛选评价图。
具体实施方式
下面的实施例将对本发明予以进一步的说明,但并不因此而限制本发明。
实施例1.人源UGT酶代谢麦冬高异黄酮A的广谱性研究
参与广谱型底物麦冬高异黄酮A葡萄糖结合代谢的UGT酶表型研究。采用13种商业化的重组人源UGT单酶(UGT1A1,UGT1A3,UGT1A4,UGT1A6,UGT1A7,UGT1A8,UGT1A9,UGT1A10,UGT2B4,UGT2B7,UGT2B10,UGT2B15,UGT2B17)作为目标UGT酶源,对底物MOA进行代谢表征。具体如下:
(1)将Tris-HCl缓冲液(50mM,pH=7.4)与氯化镁(5mM),分别以13种UGT单酶中的一个为目标UGT酶(0.05mg/ml的重组人源UGT单酶),麦冬高异黄酮A(浓度统一为10μM和100μM)在EP管中混合,于20-60℃条件下,预孵育3-5分钟,孵育体系pH值为5-10。具体反应条件如下:
UGT1A1 | UGT1A3 | UGT1A4 | UGT1A6 | UGT1A7 | UGT1A8 | UGT1A9 | |
预孵育时间(分钟) | 3 | 4 | 5 | 3 | 4 | 5 | 3 |
温度℃ | 20 | 50 | 60 | 20 | 37 | 60 | 37 |
pH值 | 5 | 8 | 10 | 6 | 9 | 7.4 | 7.4 |
UGT1A10 | UGT2B4 | UGT2B7 | UGT2B10 | UGT2B15 | UGT2B17 | ||
预孵育时间(分钟) | 4 | 5 | 3 | 4 | 5 | 5 | |
温度 | 37 | 37 | 37 | 37 | 37 | 37 | |
pH值 | 7.4 | 7.4 | 7.4 | 7.4 | 7.4 | 7.4 |
(2)分别加入UDPGA反应10-60分钟,具体如下:
UGT1A1 | UGT1A3 | UGT1A4 | UGT1A6 | UGT1A7 | UGT1A8 | UGT1A9 | |
反应时间(分钟) | 10 | 40 | 60 | 20 | 40 | 60 | 30 |
UGT1A10 | UGT2B4 | UGT2B7 | UGT2B10 | UGT2B15 | UGT2B17 | ||
反应时间(分钟) | 60 | 60 | 60 | 60 | 60 | 60 |
运用液相-紫外***检测其葡萄糖醛酸产物的生成量,并经过数据处理得到相应的底物反应速率。结果发现底物麦冬高异黄酮A能被13种UGT酶代谢。(如图1所示)
结果表明:相较于传统底物4-甲基伞形酮,麦冬高异黄酮A能被所有13种参与人体药物代谢的UGT亚型酶代谢(包括4-甲基伞形酮所不能检测的UGT1A4和UGT2B10)。基于麦冬高异黄酮A葡萄糖醛酸化反应的发现,以及建立的该检测方法所覆盖酶的数量多,可在同一条件下实现对人体中所有13种参与药物代谢的UGT酶活性进行检测,其广谱性甚至比以4-甲基伞形酮为底物的传统方法还要优越。
实施例2.麦冬高异黄酮A被UGT酶代谢产物的制备与鉴定
在运用高分辨质谱对UGT催化的麦冬高异黄酮A代谢产物进行分析鉴定之后(如图2),分别运用生物制备法和核磁共振技术对其产物MOAG进行了大量制备和结构表征。(如图6所示)。具体步骤如下:
(1)将麦冬高异黄酮A(8.52mg),Tris-HCl缓冲液(50mM,pH 7.4),MgCl2(5mM),聚氧乙烯十六烷基醚(0.1mg/mg protein)和重组表达的13种UGT酶(每种酶取1.85μL,蛋白终浓度为0.8mg/ml)混合。在37℃下预孵育5分钟后,加入UDPGA(2mM)开始反应。
(2)在37℃下孵育6小时后,通过加入冰冷的甲醇(150ml)终止反应。在20,000×g,4℃下离心20分钟后,收集上清液,然后蒸发,并使用反相柱通过制备型反向液相色谱***纯化沉淀。
(3)最后,将获得的纯化的MOAG(8.0mg,通过液相-紫外***测定纯度大于98%)以及MOA溶解在氘代甲醇中,以使用Bruker 400核磁共振(NMR)光谱仪进行结构表征。
(4)所得产物运用于方法构建中的标曲建立及相关方法评价实验。
结果表明:广谱底物麦冬高异黄酮A的葡萄糖醛酸化反应可被多种UGT酶催化代谢生成单一代谢产物单葡萄糖醛酸产物:MOA-7-O-葡萄糖醛酸化产物(MOAG),反应如下:
实施例3.稳定性考察
(1)在生理条件下将Tris-HCl缓冲液(50mM,pH=7.4)与MgCl2(5mM),以重组表达的13种UGT酶(每种酶取0.31μL,蛋白终浓度为0.1mg/ml)为酶源,底物麦冬高异黄酮A溶液(浓度选择1/10~1Km)在离心管中混合,反应温度为37℃,孵育体系pH为7.4。
(2)预孵育,将(1)中的混合液在37℃条件下孵育3~5min,保证酶与底物麦冬高异黄酮A充分接触。
(3)加入UDPGA反应时间30分钟。
(4)分别在4℃条件下放置0小时,24小时和48小时后取样分析,考察其在4℃冰箱储存条件下的稳定性。其结果如图3所示。
结果说明:稳定性强,该探针反应中,产物与底物不会相互反应,不会相互干扰,在4℃条件下利用该方法进行实验样本处理与测定,至少两天不会有明显响应信号强度变化,方便实时定量监测其葡萄糖醛酸化反应过程。
实施例4.LC-UV法检测MOA-7-O-葡萄糖醛酸化反应速率
(1)在生理条件下将Tris-HCl缓冲液(50mM,pH=7.4)与MgCl2(5mM),以重组表达的13种UGT酶(每种酶取0.31μL,蛋白终浓度0.1mg/ml)为酶源,底物麦冬高异黄酮A溶液(浓度选择1/10~1Km)在离心管中混合,总体积200微升,反应温度37℃;孵育体系pH值为7.4。
(2)预孵育。将上述混合溶液(设置无底物麦冬高异黄酮A的空白对照组)在37℃条件下孵育5min。
(3)加入UDPGA反应时间60分钟,确保所述产物生成率或底物转化率低于20%。
(4)加入等体积200微升的冰乙腈终止反应,在离心机上以20,000×g,4℃的条件下离心20分钟。反应液离心沉淀蛋白后,借助液相-紫外检测***对所生成的底物和产物进行定量分析,在紫外波长300nm下进行检测。检测条件以ODS柱为固定相,以0.2%甲酸水(B)和乙腈(A)为流动相,0-1min,80%B,1-2min,80%-60%B;2-6min,60%-20%B;6-6.5min,20%B;6.5-7.5min,20%-80%B,运用梯度洗脱方式对底物和产物进行分离。在紫外波长300nm下对底物和产物进行分析检测(图4)。
(5)测定反应时间内麦冬高异黄酮A葡萄糖醛酸产物MOAG的生成速率并计算加入抑制剂组的反应速率相对于空白对照组反应速率的百分数作为抑制活性评估标准。
检测后发现了该LC-UV法可用于检测MOA-7-O-葡萄糖醛酸化反应速率。
实施例5.LC-MS法检测MOA-7-O-葡萄糖醛酸化反应速率
(1)在生理条件下将Tris-HCl缓冲液(50mM,pH=7.4)与MgCl2(5mM),以重组表达的13种UGT酶(每种酶取0.31μL,蛋白终终浓度0.1mg/ml)为酶源,底物麦冬高异黄酮A溶液(浓度选择1/10~1Km)在离心管中混合,总体积100微升,反应温度37℃;孵育体系pH值为7.4。
(2)预孵育。将上述混合溶液(设置无底物麦冬高异黄酮A的空白对照组)在37℃条件下孵育3~5min。
(3)加入UDPGA反应时间60分钟,确保所述产物生成率或底物转化率低于20%。
(4)加入含有内标4-甲基伞型酮-beta-D-葡糖苷酸的等体积冰乙腈终止反应,在离心机上以20,000×g,4℃的条件下离心20分钟。反应液离心沉淀蛋白后,借助液相-质谱检测***对所生成的产物进行定量分析。
(5)检测条件以ODS柱为固定相,以0.2%甲酸水(B)和乙腈(A)为流动相,流速设置为0.4ml/min,进样量设置为2μL,采用梯度洗脱方式对底物和产物进行分离:0-1min,80%B,1-2min,80%-60%B;2-6min,60%-20%B;6-6.5min,20%B;6.5-7.5min,20%-80%B。采用电喷雾离子源、负离子、多反应监测(multiple reaction monitoring,MRM)模式。氮气为雾化气和干燥气;高纯氮气为碰撞气,压力是0.1MPa;质谱仪器参数设置如下:离子化电压(Is):-4500V;喷撞气(CAD):Medium;气帘气(CUR):20psi,喷雾气(GS1):20psi,离子源温度(TEM):450℃,去簇电压(DP):-80V,碰撞电压(CE):15V。底物母离子质荷比(m/z):341.105,底物子离子质荷比(m/z):206.060。产物母离子质荷比(m/z):517.138,产物子离子质荷比(m/z):341.105,175.027。检测结果如图5所示。
(6)测定反应时间内葡萄糖醛酸产物的生成速率并计算加入抑制剂组的反应速率相对于空白对照组反应速率的百分数作为抑制活性评估标准。
检测后发现了该LC-MS法可用于检测MOA-7-O-葡萄糖醛酸化反应速率。
实施例6.运用LC-UV方法对UGT酶抑制剂进行筛选与评价
非用于疾病诊断治疗的UGT酶活性的检测方法在筛选和评价UGT酶调控剂中的应用,可以筛选与评价未知化合物对13种UGT酶的抑制效果。具体操作如下:
(1)设置200μL的反应体系,将Tris-HCl缓冲液(50mM,pH=7.4),氯化镁(5mM),分别用13种重组人源UGT酶(终浓度0.05mg/ml)和麦冬高异黄酮A溶液(10μM),以及待测化合物穗花杉双黄酮、尼罗替尼、厚朴酚和氟康唑的其中一种(浓度1μM-100μM)等溶液加入离心管中,混匀,于37℃条件下预孵3分钟。
(2)向反应体系中加入10μL UDPGA(终浓度2mM)起始反应后,将离心管放入孵育器连续反应60分钟,确保所述产物生成率或底物转化率低于20%。
(3)加入等体积200μL的冰乙腈终止反应,在离心机上以20,000×g,4℃的条件下离心20分钟。反应液离心沉淀蛋白后,借助液相-紫外检测***对所生成的产物进行定量分析,按上述检测条件(实例3)在紫外波长300nm下进行检测。定量测定反应时间内葡萄糖醛酸产物的生成速率并计算加入抑制剂组的反应速率相对于空白对照组产物生成速率的百分数作为抑制活性评估标准,发现了四种化合物对UGT酶均有不同程度的抑制效果(图7)。以UGT1A1为例,加入抑制剂前,反应速率为23.97μmol/min/mg protein,而在加入穗花杉双黄酮、尼罗替尼、厚朴酚和氟康唑之后,反应速率变为1.81、2.63、13.74、21.06μmol/min/mgprotein。
实施例7.运用LC-MS方法对UGT酶抑制剂进行筛选与评价
非用于疾病诊断治疗的UGT酶活性的检测方法在筛选和评价UGT酶调控剂中的应用,可以筛选与评价未知化合物对13种UGT酶的抑制效果。具体操作如下:
(1)设置100μL的反应体系,将Tris-HCl缓冲液(50mM,pH=7.4),氯化镁(5mM),分别用13种重组人源UGT酶(终浓度0.05mg/ml)和麦冬高异黄酮A溶液(10μM),以及待测化合物穗花杉双黄酮、尼罗替尼、厚朴酚和氟康唑的其中一种(浓度1μM-100μM)等溶液加入离心管中,混匀,于37℃条件下预孵3分钟。
(2)向反应体系中加入5μL UDPGA(终浓度2mM)起始反应后,将离心管放入孵育器连续反应60分钟,确保所述产物生成率或底物转化率低于20%。
(3)加入含有内标4-甲基伞型酮-beta-D-葡糖苷酸的等体积100μL的冰乙腈终止反应,在离心机上以20,000×g,4℃的条件下离心20分钟。反应液离心沉淀蛋白后,借助液相-质谱检测***在多反应监控(MRM)模式下对所生成的产物进行定量检测。(图8)
(4)检测条件以ODS柱为固定相,以0.2%甲酸水(B)和乙腈(A)为流动相,流速设置为0.4ml/min,进样量设置为2μL,采用梯度洗脱方式对底物和产物进行分离:0-0.5min,98%B,0.5-0.6min,98%-15%B;0.6-1.6min,15%B;1.6-1.7min,98%B;1.7-3min,98%B。采用电喷雾离子源、负离子、多反应监测(multiple reaction monitoring,MRM)模式。氮气为雾化气和干燥气;高纯氮气为碰撞气,压力是0.1MPa;质谱仪器参数设置如下:离子化电压(Is):-4500V;喷撞气(CAD):Medium;气帘气(CUR):20psi,喷雾气(GS1):20psi,离子源温度(TEM):450℃,去簇电压(DP):-80V,碰撞电压(CE):15V。用于定量分析的检测离子为:MOAG质荷比517.138→341.105。内标4-甲基伞型酮-beta-D-葡糖苷酸质荷比351.0→175.0。
(5)将定量测定反应时间内葡萄糖醛酸产物的生成速率并计算加入抑制剂组的反应速率相对于空白对照组产物生成速率的百分数作为抑制活性评估标准,结果与UV检测相似,发现了四种化合物对UGT酶均有不同程度的抑制效果。以UGT1A1为例,加入抑制剂前,反应速率为22.88μmol/min/mg protein,而在加入穗花杉双黄酮、尼罗替尼、厚朴酚和氟康唑之后,反应速率变为1.62、2.31、14.20、21.97μmol/min/mg protein。
(6)该检测方法可精准定量目标产物MOAG,避免了复杂样品运用LC-UV检测时因分离效果不佳而产生的杂质峰对目标峰的干扰,且其分析时间仅需要3min,较为高效。
实施例8.运用LC-UV方法对UGT酶激活剂进行筛选与评价
非用于疾病诊断治疗的UGT酶活性的检测方法在筛选和评价UGT酶调控剂中的应用。具体操作如下:
(1)设置200μL的反应体系,将Tris-HCl缓冲液(50mM,pH=7.4),氯化镁(5mM),重组人源UGT1A1酶(终浓度0.05mg/ml)和麦冬高异黄酮A溶液(10μM),以及待测化合物黄豆黄素(浓度0μM-10μM)溶液加入离心管中,混匀,于37℃条件下预孵3分钟。
(2)向反应体系中加入10μL UDPGA(终浓度2mM)起始反应后,将离心管放入孵育器连续反应60分钟,确保所述产物生成率或底物转化率低于20%。
(3)加入等体积200μL的冰乙腈终止反应,在离心机上以20,000×g,4℃的条件下离心20分钟。反应液离心沉淀蛋白后,借助液相-紫外检测***对所生成的产物进行定量分析,按上述检测条件(和实例4一样)在紫外波长300nm下进行检测。定量测定反应时间内葡萄糖醛酸产物的生成速率并计算加入激活剂组的反应速率相对于空白对照组产物生成速率的百分数作为激活活性评估标准,发现了待测化合物黄豆黄素在10μM浓度下对UGT酶比较理想的激活效果(大于2倍)。(图9)。
Claims (6)
1.一种非用于疾病诊断治疗的UGT酶活性的检测方法,其特征在于,采用麦冬异黄酮A作为底物,通过定量检测麦冬异黄酮A的转化速率或其7-O-葡萄糖醛酸产物的生成速率来检测UGT酶的活性;
所述方法包括如下步骤:
(1)将三羟甲基氨基甲烷-盐酸缓冲液与MgCl2、麦冬高异黄酮A、UGT酶混合,预孵育3-5分钟,孵育温度为20-60℃,反应液pH值为5-10;
(2)在步骤(1)所得的反应液中加入尿苷二磷酸葡萄糖醛酸,反应10-60分钟,得到麦冬高异黄酮A的7-O-葡萄糖醛酸产物;
(3)在步骤(2)所得的反应液中加入冰乙腈或冰甲醇,沉淀蛋白后离心,得到上清液;
(4)用液相-紫外***或液相-质谱***检测步骤(3)所得上清液中麦冬高异黄酮A的转化速率或麦冬高异黄酮A的7-O-葡萄糖醛酸产物的生成速率;
所述UGT酶选自UGT1A1,UGT1A3,UGT1A4,UGT1A6,UGT1A7,UGT1A8,UGT1A9,UGT1A10,UGT2B4,UGT2B7,UGT2B10,UGT2B15或UGT2B17中的一种或几种。
2.根据权利要求1所述的方法,其特征在于:当步骤(4)使用液相-紫外***时,其检测条件为,以十八烷基硅烷柱为固定相,以0.2%甲酸水(B)和乙腈(A)为流动相,采用梯度洗脱方式分离,紫外检测波长为300nm。
3.根据权利要求1所述的方法,其特征在于:当步骤(4)使用液相-质谱***时,在步骤(3)中需要加入内标4-甲基伞型酮-β-D-葡糖苷酸。
4.根据权利要求3所述的方法,其特征在于:当步骤(4)使用液相-质谱***时,其检测条件为,以十八烷基硅烷柱为固定相,以0.2%甲酸水(B)和乙腈(A)为流动相,流速设置为0.4ml/min,进样量为2μL,采用梯度洗脱方式分离:0-0.5min,98%B,0.5-0.6min,98%-15%B;0.6-1.6min,15%B;1.6-1.7min,98%B;1.7-3min,98%B;采用电喷雾离子源、负离子、多反应监测模式;氮气为雾化气和干燥气;高纯氮气为碰撞气,压力是0.1MPa;质谱仪器参数设置如下:离子化电压:-4500V;喷撞气:Medium;气帘气:20psi,喷雾气:20psi,离子源温度:450℃,去簇电压:-80V,碰撞电压:15V;用于定量分析的检测离子为:MOAG质荷比517.138→341.105;内标4-甲基伞型酮-β-D-葡糖苷酸质荷比351.0→175.0。
5.权利要求1所述的方法在筛选和评价UGT酶调控剂中的应用,所述应用还包括在步骤(1)中加入待测化合物以及在步骤(4)中对待测化合物的调控作用进行评价。
6.根据权利要求5所述的应用,其特征在于,当待测化合物使UTG酶活性上升时为激动剂;当待测化合物使UTG酶活性下降时为抑制剂。
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