戊己丸提取物不同配伍组方对大鼠CYP450酶的影响研究
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摘要
1目的
研究戊己丸提取物不同配伍组方在体内对大鼠CYP450同工酶——CYP1A2、CYP3A1/3A2、CYP2A6、CYP2D6、CYP2E1酶活性的影响,在体外对CYP1A2、3A1/3A2、2A6、2C19、2D6、2E1酶活性的影响,从中药配伍与代谢酶关系方面探讨戊己丸配伍机理。
2方法
2.1戊己丸体外抑制大鼠肝微粒体CYP450酶活性的分析方法
戊己丸由黄连、吴茱萸及白芍组成,戊己丸配伍采用L9(34)正交表设计9个戊己丸提取物不同配伍组方,3个单方。
本研究表明,戊己丸提取物不同配伍组方及黄连在体外能显著抑制大鼠肝微粒体CYP450酶活性,吴茱萸及白芍抑制CYP450酶活性能力较弱。
2.1.1大鼠肝微粒体混合酶系
Wistar雄性成年大鼠,腹腔注射总剂量为210mg·kg-1的苯巴比妥钠及总剂量为160mg·kg-1的β-萘黄酮,大鼠经苯巴比妥钠及β-萘黄酮诱导CYP450酶表达及活性增加后,摘取大鼠肝脏,高速离心法制备肝微粒体,以KCl缓冲液混悬分装,-80℃保存备用。临用时肝微粒体冰浴低温解冻复苏,添加MgCl2、KCl、6-β-葡萄糖、氧化型辅酶Ⅱ等缓冲盐溶液及CYP450酶氧化还原反应所需的呼吸链递氢／电子载体,模拟体内细胞外液环境配制大鼠肝微粒体混合酶系溶液。
为测定药物对CYP450同工酶活性的影响,需要将一定量的探针药(待测CYP450同工酶的专属性底物)、肝微粒体混合酶系溶液以及受试药加入同一试管进行孵育反应,反应终了通过测定探针药及代谢产物的浓度,与对照组比较,可以间接反应出该CYP450同工酶活性诱导或抑制情况。
2.1.2CYP1A2酶活性分析方法
以非那西丁为CYP1A2探针药,戊己丸每一受试药组设7个梯度浓度及溶剂对照,体外孵育管内加入受试药、非那西丁、肝微粒体混合酶系后置37℃水浴中,孵育管上面覆盖保鲜膜防止溶液挥发,孵育3h后,加入0.5mL冰冷甲醇终止反应,转入1.5mL离心管,4℃冰箱放置3h沉淀蛋白后,18000r·min-1离心20min,取上清液20μL, HPLC进样测定非那西丁及代谢产物——对乙酰氨基酚浓度,对乙酰氨基酚的生成量与CYP1A2酶活性呈正相关。
本部分实验受试药为戊己丸提取物组方及戊己丸主要成分对照品组方,除本部分外,以下所指戊己丸受试药均指戊己丸提取物组方。
非那西丁及对乙酰氨基酚的HPLC条件：
Kromasil-C18色谱柱(250mm×4.6mm,5μm);流动相A：5%乙腈、15%甲醇、80%水；流动相B：10%乙腈、60%甲醇、30%水；梯度洗脱程序：0min(100%A),8min (80%A,20%B),12min (60%A,40%B),20min (100%A);运行时间：40mmin；流速：1mL·min-1；柱温：30℃；检测波长：245nm；进样量：20μL计算加与不加受试药物的情况下,非那西丁经CYP1A2代谢产生对乙酰氨基酚的量,首先按照以下公式计算不同浓度受试药对CYP1A2酶活性的抑制率：抑制率(%)=(不加受试药对乙酰氨基酚生成量-加受试药对乙酰氨基酚生成量)／不加受试药对乙酰氨基酚生成量×100%；然后采用改良Bliss法计算IC50值。
2.1.3 CYP3A1/3A2酶活性分析方法
以睾丸酮为CYP3A1/3A2探针药,戊己丸每一受试药组设7个梯度浓度及溶剂对照,体外孵育管内加入受试药、睾丸酮、肝微粒体混合酶系后置37℃水浴中,孵育管上面覆盖保鲜膜防止溶液挥发,孵育3.5h后,加入0.5mL冰冷甲醇终止反应,转入1.5mL离心管,4℃冰箱放置3h沉淀蛋白后,18000r·min-1离心20min,取上清液20μL,HPLC进样测定睾丸酮及代谢产物——6β-羟基睾丸酮浓度,6β-羟基睾丸酮的生成量与CYP3A1/3A2酶活性呈正相关。
睾丸酮及6β-羟基睾丸酮的HPLC条件：
Kromasil-C18色谱柱(250mm×4.6mm,5μm);流动相A：10%乙腈、60%甲醇、30%水；流动相B：水；流动相C：乙腈；梯度洗脱程序：0min (65%A, 35%B),4.5 min (65%A,35%C),8min(65%A,35%B);运行时间：13min；流速：1 mL·min-1；柱温：30℃；检测波长：245 nm；进样量：20μL。
计算加与不加受试药物的情况下,睾丸酮经CYP3A1/3A2代谢产生6β-羟基睾丸酮的量,按照以下公式计算不同浓度受试药对CYP3A1/3A2酶活性的抑制率：抑制率(%)=(不加受试药6β-羟基睾丸酮生成量-加受试药6β-羟基睾丸酮生成量)／不加受试药6β-羟基睾丸酮生成量×100%；然后采用改良Bliss法计算IC50值。
2.1.4 CYP2A6、2C19、2D6、2E1酶活性的cocktail探针药物分析方法
以香豆素、美芬妥因、右美沙芬、氯唑沙宗为CYP2A6、2C19、2D6、2E1的混合cocktail探针药,戊己丸每一受试药组设7个梯度浓度及溶剂对照,体外孵育管内加入受试药、混合cocktail探针药、肝微粒体混合酶系后置37℃水浴中,孵育管上面覆盖保鲜膜防止溶液挥发,孵育3h后,加入0.5mL冰冷甲醇终止反应,转入1.5mL离心管,4℃冰箱放置3h沉淀蛋白后,18000r·min-1离心20min,取上清液2μL, LC-MS进样测定4种探针药的代谢产物——7-羟基香豆素、4-羟基美芬妥因、右啡烷及6-羟基氯唑沙宗的生成浓度,该4种代谢产物的生成量与相应的CYP450同工酶活性呈正相关。
7-羟基香豆素、4-羟基美芬妥因、右啡烷及6-羟基氯唑沙宗的LC-MS条件：
Clipeus-C8色谱柱(30mm×2.1mm,5μm);流动相A：水；流动相B：甲醇；流动相C：5mM醋酸铵；梯度洗脱程序：0min (90%A,10%B), 1.0min (90%A,10%B),2.0min(60%A,40%B).3.5min(60%A,40%B),3.51min(40%B, 60%C)、4.5min(60%B,40%C)、4.51min(40%A,60%B)、5.0min(20%A,80%B)、6.0min (20%A,80%B)、6.01min (90%A,10%B)、8.0min (90%A,10%B)；运行时间：8min；流速：0.2mL·min-1；柱温：25℃；进样量：2μL; MS:ESI离子源,Drying Gas Flow:10Lmin-1；Drying Gas Tem:350℃;正负离子模式同时检测,MRM扫描模式：7-羟基香豆素,163-189正离子；4-羟基美芬妥因,201-230负离子；右啡烷,269-233正离子；6-羟基氯唑沙宗184-184负离子。
计算加与不加受试药物的情况下,探针药经相应CYP450同工酶代谢产生7-羟基香豆素、6-羟基氯唑沙宗、4-羟基美芬妥因及右啡烷的量,按照以下公式计算不同浓度受试药对CYP450同工酶活性的抑制率：抑制率(%)=(不加受试药探针药代谢产物生成量-加受试药探针药代谢产物生成量)／不加受试药探针药代谢产物生成量×100%；然后采用改良Bliss法计算IC50值。
2.2戊己丸配伍体内对大鼠CYP450酶活性影响分析方法
2.2.1大鼠体内CYP1A2酶活性分析方法
CYP1A2底物——非那西丁大鼠灌胃给药后,HPLC测定大鼠血循环中不同时间点的非那西丁、对乙酰氨基酚(非那西丁经CYP1A2代谢后的产物)浓度变化,以非那西丁、对乙酰氨基酚在给受试药物(本论文实验为戊己丸提取物不同配伍组方)前后药动学信息参数的变化推导受试药在体内对动物的CYP1A2酶活性影响。
非那西丁、对乙酰氨基酚浓度的HPLC测定条件同体外CYP1A2酶活性分析方法。
2.2.2大鼠体内CYP3A、2A6、2D6、2E1酶活性分析方法
采用CYP3A、CYP2A6、CYP2D6、CYP2E1专属性底物——睾丸酮、香豆素、右美沙芬及氯唑沙宗混合cocktail探针药物大鼠灌胃给药后,LC-MS测定大鼠血循环中不同时间点的睾丸酮／6-羟基睾丸酮(代谢产物)、香豆素／7-羟基香豆素(代谢产物)、右美沙芬／右啡烷(代谢产物)及氯唑沙宗／6-羟基氯唑沙宗(代谢产物)浓度变化,以上述探针药及代谢产物药对在给受试药物(本论文实验为戊己丸提取物不同配伍组方)前后药动学信息参数的变化推导受试药在体内对动物CYP3A、CYP2A6、CYP2D6、CYP2E1酶活性的影响。
上述探针药及代谢产物的LC-MS条件：
Kromasil-C18色谱柱(250mm×4.6mm,5μm);流动相A：甲醇；流动相B：5mmol/L醋酸铵；梯度洗脱程序：0min(28%A,72%B),5min (28%A,72%B), 7min (50%A,50%B),20min (65%A,35%B),30min (90%A，10%B);运行时间：35min；流速：1mL·min-1(柱后分流1：2,实际流量为1／3流速)；柱温：35℃；进样量：15μL。MS:API-ES离子源,除6-羟基氯唑沙宗采用负离子扫描模式,其余7待测物质(睾丸酮／6-羟基睾丸酮、香豆素／7-羟基香豆素、右美沙芬／右啡烷及氯唑沙宗)均为正离子扫描,drying gas流速10L/min, Nebulizer Pressure 40Psi, drying gas温度350℃, capillary voltage正4000负4000, Fragmentor 70ev,睾丸酮正离子扫描-289.2,氯唑沙宗-187.1,香豆素-147.1,右美沙芬-272.1,6-羟基睾丸酮-305.2,6-羟基氯唑沙宗-184.1,7-羟基香豆素-163.1,右啡烷-258.1。
2.3数据统计
实验数据采用正交t值法、SPSS 11.5. kinetica4.4软件综合分析。
3结果
3.1大鼠体外肝微粒体CYP450酶活性
大鼠经苯巴比妥钠及β-萘黄酮诱导后,CYP450酶量及活性能够满足体外研究所需,以小牛血清白蛋白为标准品,采用BCA试剂盒法测定微粒体蛋白含量,蛋白浓度为1.2g·L-1。
3.2戊己丸提取物不同配伍组方体外对大鼠肝微粒体CYP1A2酶活性抑制结果
黄连、吴茱萸、白芍提取物及1-9#方抑制CYP1A2的IC50分别为：28.07、989.69、6633.28、57.92、104.38、321.28、32.17、80.09、71.47、76.76、40.41、29.45μg(生药)·mL-1；盐酸小檗碱、吴茱萸碱及Ⅰ-Ⅸ#方(以黄连主要成分盐酸小檗碱、吴茱萸主要成分吴茱萸碱及白芍主要成分芍药苷按提取物组方中相应含量模拟戊己丸提取物正交设计的9个配比方)抑制CYP1A2的IC50分别为：5.878、6.854、8.158、11.541、12.479、10.449、6.783、5.158、6.252、4.263、3.929μg·mL-1,芍药苷对CYP1A2酶活性无影响。
戊己丸及黄连(或盐酸小檗碱)可以显著抑制CYP1A2酶活性,戊己丸提取物不同配伍组方中吴茱萸和白芍配比不同可以影响方中黄连(或盐酸小檗碱)抑制CYP1A2酶活性的能力,不同配比戊己丸抑制CYP1A2酶活性能力有统计学差异,随着黄连、吴茱萸两药在戊己丸方中剂量水平的增高可以增强戊己丸提取物不同配伍组方抑制CYP1A2酶活性的能力,随着白芍在戊己丸方中剂量水平的增高可以减弱该组方抑制CYPA2酶活性的能力,正交设计中的戊己丸的3药3水平,除白芍小、中剂量水平不同不会影响戊己丸抑制CYP1A2酶活性的能力外,其它两药各水平间,及白芍中与大、小与大水平间比较,均可以显著影响戊己丸抑制CYP1A2酶活性的能力。
3.3戊己丸提取物不同配伍组方体外对大鼠肝微粒体CYP3A1/3A2酶活性抑制结果
黄连、吴茱萸、白芍各提取物及戊己丸1-9#方抑制CYP3A1/3A2的IC50分别为：38.96、871.96、15519.17、43.17、60.47、276.12、133.40、118.08、88.47、64.36、35.13、39.91μg(生药)·mL-1；黄连及戊己丸均可显著抑制CYP3A1/3A2酶活性,不同配比戊己丸抑制CYP3A1/3A2酶活性作用不同,有统计学差异；随着黄连在戊己丸方中剂量水平的增高,戊己丸提取物不同配伍组方抑制CYP3A1/3A2酶活性的能力增强,且戊己丸提取物不同配伍组方中吴茱萸和白芍配比不同可以影响方中黄连抑制CYP3A1/3A2酶活性的程度。
3.4戊己丸提取物不同配伍组方体外对CYP2A6、2C19、2D6及2E1酶活性抑制结果
黄连、吴茱萸、白芍各提取物及戊己丸1-9#方抑制CYP2A6的IC50分别为：251.17、1925.82、2.18e+09、735.93、380.84、1271.31、293.46、490.71、283.30、596.02、228.63、422.68μg(生药)·mL-1(1-9#方IC50为黄连拆分IC50值)；抑制CYP2C19 IC50分别为：343.37、1713.23、20137.80、339.31、240.80、245.52、136.59、192.45、277.41、174.95、128.18、133.19嵋(生药)·mL-1(1-9#方IC50为黄连拆分IC50值)；抑制CYP2D6 IC50分别为：145.77、1487.94、2.84e+08、144.16、156.53、301.13、160.06、275.46、131.51、249.98、135.88、151.23μg(生药)·mL-1(1-9#方IC50为黄连拆分IC50值)；抑制CYP2E1 IC50分别为：148.67、1461.11、8.02e+31、127.23、131.66、221.22、95.51、177.27、79.50、155.20、101.69、91.47μg(生药)·mL-1(1-9#方IC50为黄连拆分IC50值)。
黄连及戊己丸均可显著抑制以上4种同工酶活性,不同配比戊己丸抑制同工酶活性作用不同,有统计学差异；戊己丸复方中各因素、水平间比较,差异较大。
3.5戊己丸提取物不同配伍组方体内对CYP1A2酶活性影响结果
对乙酰氨基酚生成量的组间协方差、多因素方差分析等统计表明,黄连零水平(对照组或吴茱萸、白芍单味药组)与大剂量水平比较,对CYP1A2酶活性影响具有显著性差异,随着黄连剂量水平的增大,酶活性呈增高趋势；吴茱萸大剂量水平与小、中剂量两水平比较,对CYP1A2酶活性影响具有显著性差异,也即戊己丸复方中引入小、中剂量水平的吴茱萸可以增高CYP1A2酶活性,但高剂量吴茱萸对CYP1A2酶活性有抑制作用；白芍零剂量与小剂量水平间、中剂量与大剂量水平间比较,对CYP1A2酶活性影响具有显著性差异,也即戊己丸复方中引入小、中剂量水平的白芍可以增高CYP1A2酶活性,但高剂量白芍对CYP1A2酶活性有抑制作用。戊己丸各因素、水平均可能对CYP1A2酶活性造成影响,各因素水平交互作用造成了戊己丸提取物不同配伍组方对CYP1A2酶活性的影响不同。
非那西丁的药动学参数统计分析表明,8#方的MRT显著低于其他各组,5#方AUC及MRT值偏高,各组间非那西丁的部分药动学参数比较,也存在差异,这表明戊己丸各因素、水平可能造成非那西丁在给药动物体内的药动学过程差异。
综合考察探针药药动过程及探针药代谢产物生成情况：随着黄连剂量比例的增加,该戊己丸受试药组动物体内CYP1A2酶活性呈增加趋势,小、中剂量吴茱萸及白芍可以增加CYP1A2酶活性,但大剂量吴茱萸及白芍可以抑制CYP1A2酶活性。
3.6戊己丸提取物不同配伍组方体内对CYP3A、2A6、2D6及2E1酶活性影响结果
戊己丸给药对香豆素在动物体内的药动过程影响显著,进一步对香豆素AUC、MRT参数进行单因素／多因素方差分析,表明戊己丸3因素(3组成味药)对香豆素AUC的影响,吴茱萸因素>白芍因素>黄连因素；在戊己丸提取物不同配伍组方中引入各剂量水平的黄连均能显著减少受试动物香豆素的体内滞留时间,戊己丸3因素对香豆素体内滞留时间的影响,白芍因素>吴茱萸因素>黄连因素。
右美沙芬药动学参数与对照组比,吴茱萸组、白芍组及1#方组显著性减少,进一步对右美沙芬AUC参数进行单因素／多因素方差分析,戊己丸3因素对右美沙芬AUC的影响,黄连因素>白芍因素>吴茱萸因素。
不同时间点各受试药组动物6-羟基氯唑沙宗生成情况组间比较,差异显著,吴茱萸零剂量与中剂量水平间有显著性差异,中剂量与大剂量水平间有显著性差异,白芍大剂量与零剂量,小,中剂量水平间有显著性差异。
不同时间点各组7-羟基香豆素生成情况组间比较.,差异显著,吴茱萸零剂量与中剂量水平间有显著性差异,中剂量与大剂量水平间有显著性差异,白芍大剂量与零剂量,小,中剂量水平间有显著性差异。
不同时间点各组右啡烷生成情况组间比较,差异显著,黄连中剂量水平与零剂量、小剂量水平间有显著性差异,吴茱萸小剂量与大剂量水平间有显著性差异,白芍小剂量与中、大剂量水平间有显著性差异。
综合考察探针药药动过程及探针药代谢产物生成情况：黄连、吴茱萸及白芍剂量比例不同可以造成组方影响CYP450酶活性的不同作用,各组动物CYP3A1／3A2酶活性大小依次为：对照组>白芍组>黄连组>7#方>3#方>6#方>5#方>吴茱萸组>8#方>2#方>4#方>9#方>1#方；各组动物CYP2E1酶活性大小依次为：7#>吴茱萸>白芍>9#＞1#>8#＞6#>3#>黄连>对照>2#>5#>4#；各组动物CYP2A6酶活性大小依次为：5#>7#>黄连>8#>9#>4#>3#＞1#＞白芍>2#>对照>6#>吴茱萸；各组动物CYP2D6酶活性大小依次为：8#>对照组>7#＞6#>2#＞3#＞4#>5#>9#＞吴茱萸>白芍>黄连＞1#。
4结论
戊己丸配比不同,对CYP450酶活性的影响作用不同,这种差异可能是不同配比戊己丸药效学、药动学差异的原因所在。
AIM
Wuji Pill is a prescription of TCM and was composed of Rhizoma Coptidis, Fructus Evodiae Rutaecarpae and Radix Paeoniae Alba. The aim of this research is to investigate the effects of Wuji Pill compound with different compatibility on the levels of enzymic activity of Cytochrome P450 in rat, and to confirm the compatibility mechanism of Wuji Pill from the point of relationships between Compound Prescription of TCM and Metabolism.
METHODS
1 Analytical method of CYP450 activity of rat Liver microsomes in vitro
Administration of wuji Pill was divided to 9 combined matching drug and 3 single drug by orthogonal design L9(34).
This study shows that different matching ratio of Wuji Pill and coptis chinensis can inhibit activity of rat liver microsomes CYP450 in vitro significantly, and this inhibition of Fructus Evodiae Rutaecarpae and Radix Paeoniae Alba on CYP450 is weak.
1.1 Preparation of combination enzymes of Rat liver microsomes
Wistar male rats, after intraperitoneal injection of a total dose of 210 30mg·kg-1 of phenobarbital sodium and total dose of 160mg·kg-1 ofβ-naphthoflavone to induce increased expression and activity of CYP450 enzymes, were took out rat liver to preparation of combination enzymes of Rat liver microsomes.
For the effect determination of drugs on the activity of CYP450 isoenzymes, a certain amount of probe drugs, liver microsomal enzyme and tested drugs were incubated in vitro. Concentration of probe drugs and metabolites were measured in the end of reaction. Compare with the control group, the activity of CYP450 isoenzyme can be calculated indirectedly.
1.2 Analytical method of CYP1A2 activity of rat Liver microsomes in vitro
With Phenacetin being a probe, the levels of enzymic activity of CYP1A2 were detected by HPLC, which were suppressed by Wuji Pill with different compatibility in vitro.
This part of the experiment subjects were Wuji pill extracts and main components of reference substance. Except this section, the following experiment subjects were meaning extracts alone.
HPLC conditions of Phenacetin and paracetamol:
Kromasil-C18 column (250mm×4.6mm,5μm); mobile phase A:5%acetonitrile, 15%methanol,80%water; mobile phase B:10%acetonitrile,60%methanol,30% water; gradient elution program:Omin (100%A),8min (80%A,20%B),12min (60%A,40%B),20min (100%A); running time:40min; flow rate:1mL·min-1; column temperature:30℃; detection wavelength:245nm; injection volume:20μL.
1.3 Analytical method of CYP3A1/3A2 activity of rat Liver microsomes in vitro
With Testosterone being a probe, the levels of enzymic activity of CYP3A1/3A2 were detected by HPLC, which were suppressed by Wuji Pill with different compatibility in vitro.
HPLC conditions of Testosterone and 6β-hydroxy testosterone:
Kromasil-Ci8 column (250 mm×4.6 mm,5μm); mobile phase A:10% acetonitrile,60%methanol,30%water; mobile phase B:water; mobile phase C: acetonitrile; gradient elution program:0 min (65%A,35%B),4.5 min (65%A,35% C),8 min (65%A,35%B); running time:13 min; flow rate:1 mL·min-1; column temperature:30℃; detection wavelength:245 nm; injection volume:20μL.
1.4 Analytical method of CYP2A6,2C19,2D6,2E1 activity of rat Liver microsomes in vitro
With coumarin, Mephenetoin, Dextromethorphan, chlorzoxazone being a cocktail probe, the levels of enzymic activity of CYP450 isoenzymes above were detected by LC-MS, which were suppressed by Wuji Pill with different compatibility in vitro.
LC-MS conditions of cocktail probe:
Clipeus-C8 column (30mm×2.1mm,5μm); mobile phase A:Water; mobile phase B:methanol; mobile phase C:5mM ammonium acetate; gradient elution program: Omin (90%A,10%B), 1.0min (90%A,10%B),2.0min (60%A,40%B),3.5min (60%A,40%B),3.51min (40%B,60%C),4.5min (60%B,40%C),4.51min (40% A,60%B),5.0min (20%A,80%B),6.0min (20%A,80%B),6.01min (90%A, 10%B),8.0min (90%A,10%B); running time:8min; flow rate:0.2mL·min-1; column temperature:25℃; injection volume:2μL; MS:ESI ion Source, Drying Gas Flow:10L·min-1; Drying Gas Tem:350℃; positive and negative mode while testing, MRM scan mode:7-hydroxycoumarin,163-189 positive ions; 4-hydroxy Meifen properly due,201-230 negative ions; the right of non-methane,269-233 positive ions; 6-hydroxy-chlorzoxazone 184-184 negative ions.
2 Analytical method of CYP450 activity of rat Liver microsomes in vivo
2.2 Analytical method of CYP1A2 activity of rat Liver microsomes in vivo
CYP1A2 substrate-phenacetin after oral administration in rats, HPLC determination of blood drug concentration in rats at different time points of phenacetin. The change of the pharmacokinetic parameters information may represent change of CYP450 activity.
HPLC conditions of Phenacetin, paracetamol as in vitro analysis.
2.2 Analytical method of CYP3A,2A6,2D6,2E1 activity of rat Liver microsomes in vivo
With testosterone, coumarin, Dextromethorphan, chlorzoxazone being a cocktail probe in vivo, the LC-MS conditions of cocktail probe:
Kromasil-C18 column (250mm×4.6mm,5μm); mobile phase A:methanol; mobile phase B:5mmol/L ammonium acetate; gradient elution program:Omin (28%A,72%B),5min (28%A,72%B),7min (50%A,50%B),20min (65%A, 35%B),30min (90%A,10%B); running time:35min; flow rate:1mL·min-1 (1:2 segregation after column, the actual flow rate of 1/3 flow); column temperature:35℃; injection volume:15μL. MS:API-ES ion source, in addition to 6-hydroxy-chlorzoxazone in negative scan mode, the remaining 7 tested substances (testosterone/6-hydroxy-testosterone, coumarin/7-hydroxy coumarin, dextrome-thorphan/right non-alkane and chlorzoxazone) are positive ion scan, drying gas velocity lOL/min, Nebulizer Pressure 40Psi, drying gas temperature of 350℃, capillary voltage 4000 is negative 4000, Fragmentor 70ev, testosterone positive ion scan-289.2, chlorineylsand were-187.1,-147.1 coumarin, dextromethorphan,-272.1,6-hydroxy-testosterone-305.2,6-hydroxy chlorzoxazone-184.1,7-hydroxyl-coumarin-163.1, right non-alky 1-258.1.
3 Statistics
Experimental data were statistical analysis by T value analysis of orthogonality, SPSS 11.5 and kinetica4.4 software.
RESULTS
1 Enzymic activity of rat liver microsomes
Rats after inducted by phenobarbital andβ-naphthoflavone, CYP450 enzyme concentration and activity in vitro can to meet the requirements of experiments. Determined by BCA kit microsomal protein content, protein concentration is 1.2g·L-1.
2 Effect of Wuji Pill on CYP1A2 activity of rat Liver microsomes in vitro
The IC50 of Rhizoma Coptidis, Fructus Evodiae Rutaecarpae, Radix Paeoniae Alba and 1-9# of different level Wuji Pill is:28.07,989.69,6633.28,57.92,104.38, 321.28,32.17,80.09,71.47,76.76,40.41 and 29.45μg(crude drug)/mL, respectively; Rhizoma Coptidis and 1-9 of Wuji Pill can suppress the enzymic activity of CYP1A2 significantly, and the capability of Rhizoma Coptidis in Wuji Pill of action on CYP1A2 can be modified by different composition of Fructus Evodiae Rutaecarpae and Radix Paeoniae Alba in Wuji Pill, and there are statistical difference among the IC50 of 1-9# of Wuji Pill; while the ratio of Rhizoma Coptidis and Fructus Evodiae Rutaecarpae raising up in Wuji Pill, Wuji Pill may supperess the enzymic of CYP1A2 strengthenly and with the ratio of Radix Paeoniae Alba raising up in Wuji Pill, the suppressed capability of Wuji Pill on CYP1A2 should be weaken oppositely.
3 Effect of Wuji Pill on CYP3A1/3A2 activity of rat Liver microsomes in vitro
The IC50 of Rhizoma Coptidis, Fructus Evodiae Rutaecarpae, Radix Paeoniae Alba and 1-9# of different level Wuji Pill is:38.96,871.96,15519.17,43.17,60.47, 276.12,133.40,118.08,88.47,64.36,35.13 and 39.91μg (crude drug)·mL-1, respectively; Rhizoma Coptidis and 1-9# of Wuji Pill can suppress the enzymic activity of CYP3A1/3A2 significantly, and the capability of Rhizoma Coptidis in Wuji Pill of action on CYP3A1/3A2 can be modified by different composition of Fructus Evodiae Rutaecarpae and Radix Paeoniae in Wuji Pill, and there are statistical difference among the IC50 of 1-9# of Wuji Pill; while the ratio of Rhizoma Coptidis raising up in Wuji Pill, Wuji Pill may supperess the enzymic activity of CYP1A2 strengthenly. Conclusion:The reason why Wuji Pill with different compatibility has different pharmacodynamics and pharmacokinetics characteristics is likely to lie in the difference of the capability of Wuji Pill with different compatibility on CYP3A1/3A2.
3 Effect of Wuji Pill on CYP2A6,2C19,2D6 and 2E1 activity of rat Liver microsomes in vitro
The IC50 of Rhizoma Coptidis, Fructus Evodiae Rutaecarpae, Radix Paeoniae Alba and 1-9# of different level Wuji Pill to CYP2A6 is:251.17,1925.82,2.18 e +09,735.93,380.84,1271.31,293.46,490.71,283.30,596.02,228.63,422.68μg (crude drug)·mL-1 respectively.
The IC50 of Rhizoma Coptidis, Fructus Evodiae Rutaecarpae, Radix Paeoniae Alba and 1-9# of different level Wuji Pill to CYP2C19 is:343.37,1713.23,20137.80, 339.31,240.80,245.52,136.59,192.45,277.41,174.95,128.18,133.19μg (crude drug)·mL-1 respectively.
The IC50 of Rhizoma Coptidis, Fructus Evodiae Rutaecarpae, Radix Paeoniae Alba and 1-9# of different level Wuji Pill to CYP2D6 is:145.77,1487.94,2.84 e +08,144.16,156.53,301.13,160.06,275.46,131.51,249.98,135.88,151.23μg (crude drug)·mL-1 respectively.
The IC50 of Rhizoma Coptidis, Fructus Evodiae Rutaecarpae, Radix Paeoniae Alba and 1-9# of different level Wuji Pill to CYP2E1 is:148.67,1461.11,8.02 e+31, 127.23,131.66,221.22,95.51,177.27,79.50,155.20,101.69,91.47μg (crude drug)·mL-1 respectively.
Rhizoma Coptidis and 1-9# of Wuji Pill can suppress the enzymic activity of CYP450 mentioned above significantly, and the capability of Rhizoma Coptidis in Wuji Pill of action on CYP450 can be modified by different composition of Fructus Evodiae Rutaecarpae and Radix Paeoniae in Wuji Pill, and there are statistical difference among the IC50 of 1-9# of Wuji Pill.
4 Effect of Wuji Pill on CYP1A2 activity of rat in vivo
The group covariance of Paracetamol concentration, and other multivariate analysis of variance show that Rhizoma Coptidis zero level (the control group or Fructus Evodiae Rutaecarpae, Radix Paeoniae Alba single administration group) compared with the high dose level of CYP1A2 enzyme activity was significant different. With the increase of Rhizoma Coptidis, activity tends to increase; and there are statistical difference among the IC50 of 1-9# of Wuji Pill.
5 Effect of Wuji Pill on CYP3A,2A6,2D6,2E1 activity of rat in vivo
Administration of Wuji Pill can significantly affected the process of coumarin in vivo in animals, further AUC, MRT parameters of univariate/multivariate analysis of variance, showed that effect of 3 factors of Wuji Pill (3 composed of herbs) on AUC of coumarin, Fructus Evodiae Rutaecarpae factor> Radix Paeoniae Alba factor> Rhizoma Coptidis factor.
univariate/multivariate analysis of variance of Dextromethorphan pharmaco-kinetic parameters show that:the control group, Fructus Evodiae Rutaecarpae group, Radix Paeoniae Alba group and 1# group was significantly reduced, effect of Wuji Pill on AUC of dextromethorphan, Rhizoma Coptidis factor> Radix Paeoniae Alba factor> Fructus Evodiae Rutaecarpae factor.
Different time points tested drugs concentration of 6-hydroxy-chlorzoxazone among experimental groups, the difference was significant, Fructus Evodiae Rutaecarpae zero dose and the middle dose level had significant differences; between middle dose and high dose level had significant difference.
Different time points tested drugs concentration of 7-hydroxycoumarin among experimental groups, the difference was significant, Fructus Evodiae Rutaecarpae zero dose and the middle dose level had significant differences; between middle dose and high dose level had significant difference.
Different time points tested drugs concentration of Dextrorphan among experimental groups, the difference was significant, Rhizoma Coptidis middle dose level compare with zero level and small dose level, the difference was significant; Fructus Evodiae Rutaecarpae small dose level compare with high dose level, the difference was significant; Radix Paeoniae Alba small dose level compare with middle dose level and high dose level, the difference was significant.
CONCLUSION
The reason why Wuji Pill with different compatibility has different pharmacodynamics and pharmacokinetics characteristics is likely to lie in the difference of the capability of Wuji Pill with different compatibility on CYP450.
研究戊己丸提取物不同配伍组方在体内对大鼠CYP450同工酶——CYP1A2、CYP3A1/3A2、CYP2A6、CYP2D6、CYP2E1酶活性的影响,在体外对CYP1A2、3A1/3A2、2A6、2C19、2D6、2E1酶活性的影响,从中药配伍与代谢酶关系方面探讨戊己丸配伍机理。
2方法
2.1戊己丸体外抑制大鼠肝微粒体CYP450酶活性的分析方法
戊己丸由黄连、吴茱萸及白芍组成,戊己丸配伍采用L9(34)正交表设计9个戊己丸提取物不同配伍组方,3个单方。
本研究表明,戊己丸提取物不同配伍组方及黄连在体外能显著抑制大鼠肝微粒体CYP450酶活性,吴茱萸及白芍抑制CYP450酶活性能力较弱。
2.1.1大鼠肝微粒体混合酶系
Wistar雄性成年大鼠,腹腔注射总剂量为210mg·kg-1的苯巴比妥钠及总剂量为160mg·kg-1的β-萘黄酮,大鼠经苯巴比妥钠及β-萘黄酮诱导CYP450酶表达及活性增加后,摘取大鼠肝脏,高速离心法制备肝微粒体,以KCl缓冲液混悬分装,-80℃保存备用。临用时肝微粒体冰浴低温解冻复苏,添加MgCl2、KCl、6-β-葡萄糖、氧化型辅酶Ⅱ等缓冲盐溶液及CYP450酶氧化还原反应所需的呼吸链递氢／电子载体,模拟体内细胞外液环境配制大鼠肝微粒体混合酶系溶液。
为测定药物对CYP450同工酶活性的影响,需要将一定量的探针药(待测CYP450同工酶的专属性底物)、肝微粒体混合酶系溶液以及受试药加入同一试管进行孵育反应,反应终了通过测定探针药及代谢产物的浓度,与对照组比较,可以间接反应出该CYP450同工酶活性诱导或抑制情况。
2.1.2CYP1A2酶活性分析方法
以非那西丁为CYP1A2探针药,戊己丸每一受试药组设7个梯度浓度及溶剂对照,体外孵育管内加入受试药、非那西丁、肝微粒体混合酶系后置37℃水浴中,孵育管上面覆盖保鲜膜防止溶液挥发,孵育3h后,加入0.5mL冰冷甲醇终止反应,转入1.5mL离心管,4℃冰箱放置3h沉淀蛋白后,18000r·min-1离心20min,取上清液20μL, HPLC进样测定非那西丁及代谢产物——对乙酰氨基酚浓度,对乙酰氨基酚的生成量与CYP1A2酶活性呈正相关。
本部分实验受试药为戊己丸提取物组方及戊己丸主要成分对照品组方,除本部分外,以下所指戊己丸受试药均指戊己丸提取物组方。
非那西丁及对乙酰氨基酚的HPLC条件：
Kromasil-C18色谱柱(250mm×4.6mm,5μm);流动相A：5%乙腈、15%甲醇、80%水；流动相B：10%乙腈、60%甲醇、30%水；梯度洗脱程序：0min(100%A),8min (80%A,20%B),12min (60%A,40%B),20min (100%A);运行时间：40mmin；流速：1mL·min-1；柱温：30℃；检测波长：245nm；进样量：20μL计算加与不加受试药物的情况下,非那西丁经CYP1A2代谢产生对乙酰氨基酚的量,首先按照以下公式计算不同浓度受试药对CYP1A2酶活性的抑制率：抑制率(%)=(不加受试药对乙酰氨基酚生成量-加受试药对乙酰氨基酚生成量)／不加受试药对乙酰氨基酚生成量×100%；然后采用改良Bliss法计算IC50值。
2.1.3 CYP3A1/3A2酶活性分析方法
以睾丸酮为CYP3A1/3A2探针药,戊己丸每一受试药组设7个梯度浓度及溶剂对照,体外孵育管内加入受试药、睾丸酮、肝微粒体混合酶系后置37℃水浴中,孵育管上面覆盖保鲜膜防止溶液挥发,孵育3.5h后,加入0.5mL冰冷甲醇终止反应,转入1.5mL离心管,4℃冰箱放置3h沉淀蛋白后,18000r·min-1离心20min,取上清液20μL,HPLC进样测定睾丸酮及代谢产物——6β-羟基睾丸酮浓度,6β-羟基睾丸酮的生成量与CYP3A1/3A2酶活性呈正相关。
睾丸酮及6β-羟基睾丸酮的HPLC条件：
Kromasil-C18色谱柱(250mm×4.6mm,5μm);流动相A：10%乙腈、60%甲醇、30%水；流动相B：水；流动相C：乙腈；梯度洗脱程序：0min (65%A, 35%B),4.5 min (65%A,35%C),8min(65%A,35%B);运行时间：13min；流速：1 mL·min-1；柱温：30℃；检测波长：245 nm；进样量：20μL。
计算加与不加受试药物的情况下,睾丸酮经CYP3A1/3A2代谢产生6β-羟基睾丸酮的量,按照以下公式计算不同浓度受试药对CYP3A1/3A2酶活性的抑制率：抑制率(%)=(不加受试药6β-羟基睾丸酮生成量-加受试药6β-羟基睾丸酮生成量)／不加受试药6β-羟基睾丸酮生成量×100%；然后采用改良Bliss法计算IC50值。
2.1.4 CYP2A6、2C19、2D6、2E1酶活性的cocktail探针药物分析方法
以香豆素、美芬妥因、右美沙芬、氯唑沙宗为CYP2A6、2C19、2D6、2E1的混合cocktail探针药,戊己丸每一受试药组设7个梯度浓度及溶剂对照,体外孵育管内加入受试药、混合cocktail探针药、肝微粒体混合酶系后置37℃水浴中,孵育管上面覆盖保鲜膜防止溶液挥发,孵育3h后,加入0.5mL冰冷甲醇终止反应,转入1.5mL离心管,4℃冰箱放置3h沉淀蛋白后,18000r·min-1离心20min,取上清液2μL, LC-MS进样测定4种探针药的代谢产物——7-羟基香豆素、4-羟基美芬妥因、右啡烷及6-羟基氯唑沙宗的生成浓度,该4种代谢产物的生成量与相应的CYP450同工酶活性呈正相关。
7-羟基香豆素、4-羟基美芬妥因、右啡烷及6-羟基氯唑沙宗的LC-MS条件：
Clipeus-C8色谱柱(30mm×2.1mm,5μm);流动相A：水；流动相B：甲醇；流动相C：5mM醋酸铵；梯度洗脱程序：0min (90%A,10%B), 1.0min (90%A,10%B),2.0min(60%A,40%B).3.5min(60%A,40%B),3.51min(40%B, 60%C)、4.5min(60%B,40%C)、4.51min(40%A,60%B)、5.0min(20%A,80%B)、6.0min (20%A,80%B)、6.01min (90%A,10%B)、8.0min (90%A,10%B)；运行时间：8min；流速：0.2mL·min-1；柱温：25℃；进样量：2μL; MS:ESI离子源,Drying Gas Flow:10Lmin-1；Drying Gas Tem:350℃;正负离子模式同时检测,MRM扫描模式：7-羟基香豆素,163-189正离子；4-羟基美芬妥因,201-230负离子；右啡烷,269-233正离子；6-羟基氯唑沙宗184-184负离子。
计算加与不加受试药物的情况下,探针药经相应CYP450同工酶代谢产生7-羟基香豆素、6-羟基氯唑沙宗、4-羟基美芬妥因及右啡烷的量,按照以下公式计算不同浓度受试药对CYP450同工酶活性的抑制率：抑制率(%)=(不加受试药探针药代谢产物生成量-加受试药探针药代谢产物生成量)／不加受试药探针药代谢产物生成量×100%；然后采用改良Bliss法计算IC50值。
2.2戊己丸配伍体内对大鼠CYP450酶活性影响分析方法
2.2.1大鼠体内CYP1A2酶活性分析方法
CYP1A2底物——非那西丁大鼠灌胃给药后,HPLC测定大鼠血循环中不同时间点的非那西丁、对乙酰氨基酚(非那西丁经CYP1A2代谢后的产物)浓度变化,以非那西丁、对乙酰氨基酚在给受试药物(本论文实验为戊己丸提取物不同配伍组方)前后药动学信息参数的变化推导受试药在体内对动物的CYP1A2酶活性影响。
非那西丁、对乙酰氨基酚浓度的HPLC测定条件同体外CYP1A2酶活性分析方法。
2.2.2大鼠体内CYP3A、2A6、2D6、2E1酶活性分析方法
采用CYP3A、CYP2A6、CYP2D6、CYP2E1专属性底物——睾丸酮、香豆素、右美沙芬及氯唑沙宗混合cocktail探针药物大鼠灌胃给药后,LC-MS测定大鼠血循环中不同时间点的睾丸酮／6-羟基睾丸酮(代谢产物)、香豆素／7-羟基香豆素(代谢产物)、右美沙芬／右啡烷(代谢产物)及氯唑沙宗／6-羟基氯唑沙宗(代谢产物)浓度变化,以上述探针药及代谢产物药对在给受试药物(本论文实验为戊己丸提取物不同配伍组方)前后药动学信息参数的变化推导受试药在体内对动物CYP3A、CYP2A6、CYP2D6、CYP2E1酶活性的影响。
上述探针药及代谢产物的LC-MS条件：
Kromasil-C18色谱柱(250mm×4.6mm,5μm);流动相A：甲醇；流动相B：5mmol/L醋酸铵；梯度洗脱程序：0min(28%A,72%B),5min (28%A,72%B), 7min (50%A,50%B),20min (65%A,35%B),30min (90%A，10%B);运行时间：35min；流速：1mL·min-1(柱后分流1：2,实际流量为1／3流速)；柱温：35℃；进样量：15μL。MS:API-ES离子源,除6-羟基氯唑沙宗采用负离子扫描模式,其余7待测物质(睾丸酮／6-羟基睾丸酮、香豆素／7-羟基香豆素、右美沙芬／右啡烷及氯唑沙宗)均为正离子扫描,drying gas流速10L/min, Nebulizer Pressure 40Psi, drying gas温度350℃, capillary voltage正4000负4000, Fragmentor 70ev,睾丸酮正离子扫描-289.2,氯唑沙宗-187.1,香豆素-147.1,右美沙芬-272.1,6-羟基睾丸酮-305.2,6-羟基氯唑沙宗-184.1,7-羟基香豆素-163.1,右啡烷-258.1。
2.3数据统计
实验数据采用正交t值法、SPSS 11.5. kinetica4.4软件综合分析。
3结果
3.1大鼠体外肝微粒体CYP450酶活性
大鼠经苯巴比妥钠及β-萘黄酮诱导后,CYP450酶量及活性能够满足体外研究所需,以小牛血清白蛋白为标准品,采用BCA试剂盒法测定微粒体蛋白含量,蛋白浓度为1.2g·L-1。
3.2戊己丸提取物不同配伍组方体外对大鼠肝微粒体CYP1A2酶活性抑制结果
黄连、吴茱萸、白芍提取物及1-9#方抑制CYP1A2的IC50分别为：28.07、989.69、6633.28、57.92、104.38、321.28、32.17、80.09、71.47、76.76、40.41、29.45μg(生药)·mL-1；盐酸小檗碱、吴茱萸碱及Ⅰ-Ⅸ#方(以黄连主要成分盐酸小檗碱、吴茱萸主要成分吴茱萸碱及白芍主要成分芍药苷按提取物组方中相应含量模拟戊己丸提取物正交设计的9个配比方)抑制CYP1A2的IC50分别为：5.878、6.854、8.158、11.541、12.479、10.449、6.783、5.158、6.252、4.263、3.929μg·mL-1,芍药苷对CYP1A2酶活性无影响。
戊己丸及黄连(或盐酸小檗碱)可以显著抑制CYP1A2酶活性,戊己丸提取物不同配伍组方中吴茱萸和白芍配比不同可以影响方中黄连(或盐酸小檗碱)抑制CYP1A2酶活性的能力,不同配比戊己丸抑制CYP1A2酶活性能力有统计学差异,随着黄连、吴茱萸两药在戊己丸方中剂量水平的增高可以增强戊己丸提取物不同配伍组方抑制CYP1A2酶活性的能力,随着白芍在戊己丸方中剂量水平的增高可以减弱该组方抑制CYPA2酶活性的能力,正交设计中的戊己丸的3药3水平,除白芍小、中剂量水平不同不会影响戊己丸抑制CYP1A2酶活性的能力外,其它两药各水平间,及白芍中与大、小与大水平间比较,均可以显著影响戊己丸抑制CYP1A2酶活性的能力。
3.3戊己丸提取物不同配伍组方体外对大鼠肝微粒体CYP3A1/3A2酶活性抑制结果
黄连、吴茱萸、白芍各提取物及戊己丸1-9#方抑制CYP3A1/3A2的IC50分别为：38.96、871.96、15519.17、43.17、60.47、276.12、133.40、118.08、88.47、64.36、35.13、39.91μg(生药)·mL-1；黄连及戊己丸均可显著抑制CYP3A1/3A2酶活性,不同配比戊己丸抑制CYP3A1/3A2酶活性作用不同,有统计学差异；随着黄连在戊己丸方中剂量水平的增高,戊己丸提取物不同配伍组方抑制CYP3A1/3A2酶活性的能力增强,且戊己丸提取物不同配伍组方中吴茱萸和白芍配比不同可以影响方中黄连抑制CYP3A1/3A2酶活性的程度。
3.4戊己丸提取物不同配伍组方体外对CYP2A6、2C19、2D6及2E1酶活性抑制结果
黄连、吴茱萸、白芍各提取物及戊己丸1-9#方抑制CYP2A6的IC50分别为：251.17、1925.82、2.18e+09、735.93、380.84、1271.31、293.46、490.71、283.30、596.02、228.63、422.68μg(生药)·mL-1(1-9#方IC50为黄连拆分IC50值)；抑制CYP2C19 IC50分别为：343.37、1713.23、20137.80、339.31、240.80、245.52、136.59、192.45、277.41、174.95、128.18、133.19嵋(生药)·mL-1(1-9#方IC50为黄连拆分IC50值)；抑制CYP2D6 IC50分别为：145.77、1487.94、2.84e+08、144.16、156.53、301.13、160.06、275.46、131.51、249.98、135.88、151.23μg(生药)·mL-1(1-9#方IC50为黄连拆分IC50值)；抑制CYP2E1 IC50分别为：148.67、1461.11、8.02e+31、127.23、131.66、221.22、95.51、177.27、79.50、155.20、101.69、91.47μg(生药)·mL-1(1-9#方IC50为黄连拆分IC50值)。
黄连及戊己丸均可显著抑制以上4种同工酶活性,不同配比戊己丸抑制同工酶活性作用不同,有统计学差异；戊己丸复方中各因素、水平间比较,差异较大。
3.5戊己丸提取物不同配伍组方体内对CYP1A2酶活性影响结果
对乙酰氨基酚生成量的组间协方差、多因素方差分析等统计表明,黄连零水平(对照组或吴茱萸、白芍单味药组)与大剂量水平比较,对CYP1A2酶活性影响具有显著性差异,随着黄连剂量水平的增大,酶活性呈增高趋势；吴茱萸大剂量水平与小、中剂量两水平比较,对CYP1A2酶活性影响具有显著性差异,也即戊己丸复方中引入小、中剂量水平的吴茱萸可以增高CYP1A2酶活性,但高剂量吴茱萸对CYP1A2酶活性有抑制作用；白芍零剂量与小剂量水平间、中剂量与大剂量水平间比较,对CYP1A2酶活性影响具有显著性差异,也即戊己丸复方中引入小、中剂量水平的白芍可以增高CYP1A2酶活性,但高剂量白芍对CYP1A2酶活性有抑制作用。戊己丸各因素、水平均可能对CYP1A2酶活性造成影响,各因素水平交互作用造成了戊己丸提取物不同配伍组方对CYP1A2酶活性的影响不同。
非那西丁的药动学参数统计分析表明,8#方的MRT显著低于其他各组,5#方AUC及MRT值偏高,各组间非那西丁的部分药动学参数比较,也存在差异,这表明戊己丸各因素、水平可能造成非那西丁在给药动物体内的药动学过程差异。
综合考察探针药药动过程及探针药代谢产物生成情况：随着黄连剂量比例的增加,该戊己丸受试药组动物体内CYP1A2酶活性呈增加趋势,小、中剂量吴茱萸及白芍可以增加CYP1A2酶活性,但大剂量吴茱萸及白芍可以抑制CYP1A2酶活性。
3.6戊己丸提取物不同配伍组方体内对CYP3A、2A6、2D6及2E1酶活性影响结果
戊己丸给药对香豆素在动物体内的药动过程影响显著,进一步对香豆素AUC、MRT参数进行单因素／多因素方差分析,表明戊己丸3因素(3组成味药)对香豆素AUC的影响,吴茱萸因素>白芍因素>黄连因素；在戊己丸提取物不同配伍组方中引入各剂量水平的黄连均能显著减少受试动物香豆素的体内滞留时间,戊己丸3因素对香豆素体内滞留时间的影响,白芍因素>吴茱萸因素>黄连因素。
右美沙芬药动学参数与对照组比,吴茱萸组、白芍组及1#方组显著性减少,进一步对右美沙芬AUC参数进行单因素／多因素方差分析,戊己丸3因素对右美沙芬AUC的影响,黄连因素>白芍因素>吴茱萸因素。
不同时间点各受试药组动物6-羟基氯唑沙宗生成情况组间比较,差异显著,吴茱萸零剂量与中剂量水平间有显著性差异,中剂量与大剂量水平间有显著性差异,白芍大剂量与零剂量,小,中剂量水平间有显著性差异。
不同时间点各组7-羟基香豆素生成情况组间比较.,差异显著,吴茱萸零剂量与中剂量水平间有显著性差异,中剂量与大剂量水平间有显著性差异,白芍大剂量与零剂量,小,中剂量水平间有显著性差异。
不同时间点各组右啡烷生成情况组间比较,差异显著,黄连中剂量水平与零剂量、小剂量水平间有显著性差异,吴茱萸小剂量与大剂量水平间有显著性差异,白芍小剂量与中、大剂量水平间有显著性差异。
综合考察探针药药动过程及探针药代谢产物生成情况：黄连、吴茱萸及白芍剂量比例不同可以造成组方影响CYP450酶活性的不同作用,各组动物CYP3A1／3A2酶活性大小依次为：对照组>白芍组>黄连组>7#方>3#方>6#方>5#方>吴茱萸组>8#方>2#方>4#方>9#方>1#方；各组动物CYP2E1酶活性大小依次为：7#>吴茱萸>白芍>9#＞1#>8#＞6#>3#>黄连>对照>2#>5#>4#；各组动物CYP2A6酶活性大小依次为：5#>7#>黄连>8#>9#>4#>3#＞1#＞白芍>2#>对照>6#>吴茱萸；各组动物CYP2D6酶活性大小依次为：8#>对照组>7#＞6#>2#＞3#＞4#>5#>9#＞吴茱萸>白芍>黄连＞1#。
4结论
戊己丸配比不同,对CYP450酶活性的影响作用不同,这种差异可能是不同配比戊己丸药效学、药动学差异的原因所在。
AIM
Wuji Pill is a prescription of TCM and was composed of Rhizoma Coptidis, Fructus Evodiae Rutaecarpae and Radix Paeoniae Alba. The aim of this research is to investigate the effects of Wuji Pill compound with different compatibility on the levels of enzymic activity of Cytochrome P450 in rat, and to confirm the compatibility mechanism of Wuji Pill from the point of relationships between Compound Prescription of TCM and Metabolism.
METHODS
1 Analytical method of CYP450 activity of rat Liver microsomes in vitro
Administration of wuji Pill was divided to 9 combined matching drug and 3 single drug by orthogonal design L9(34).
This study shows that different matching ratio of Wuji Pill and coptis chinensis can inhibit activity of rat liver microsomes CYP450 in vitro significantly, and this inhibition of Fructus Evodiae Rutaecarpae and Radix Paeoniae Alba on CYP450 is weak.
1.1 Preparation of combination enzymes of Rat liver microsomes
Wistar male rats, after intraperitoneal injection of a total dose of 210 30mg·kg-1 of phenobarbital sodium and total dose of 160mg·kg-1 ofβ-naphthoflavone to induce increased expression and activity of CYP450 enzymes, were took out rat liver to preparation of combination enzymes of Rat liver microsomes.
For the effect determination of drugs on the activity of CYP450 isoenzymes, a certain amount of probe drugs, liver microsomal enzyme and tested drugs were incubated in vitro. Concentration of probe drugs and metabolites were measured in the end of reaction. Compare with the control group, the activity of CYP450 isoenzyme can be calculated indirectedly.
1.2 Analytical method of CYP1A2 activity of rat Liver microsomes in vitro
With Phenacetin being a probe, the levels of enzymic activity of CYP1A2 were detected by HPLC, which were suppressed by Wuji Pill with different compatibility in vitro.
This part of the experiment subjects were Wuji pill extracts and main components of reference substance. Except this section, the following experiment subjects were meaning extracts alone.
HPLC conditions of Phenacetin and paracetamol:
Kromasil-C18 column (250mm×4.6mm,5μm); mobile phase A:5%acetonitrile, 15%methanol,80%water; mobile phase B:10%acetonitrile,60%methanol,30% water; gradient elution program:Omin (100%A),8min (80%A,20%B),12min (60%A,40%B),20min (100%A); running time:40min; flow rate:1mL·min-1; column temperature:30℃; detection wavelength:245nm; injection volume:20μL.
1.3 Analytical method of CYP3A1/3A2 activity of rat Liver microsomes in vitro
With Testosterone being a probe, the levels of enzymic activity of CYP3A1/3A2 were detected by HPLC, which were suppressed by Wuji Pill with different compatibility in vitro.
HPLC conditions of Testosterone and 6β-hydroxy testosterone:
Kromasil-Ci8 column (250 mm×4.6 mm,5μm); mobile phase A:10% acetonitrile,60%methanol,30%water; mobile phase B:water; mobile phase C: acetonitrile; gradient elution program:0 min (65%A,35%B),4.5 min (65%A,35% C),8 min (65%A,35%B); running time:13 min; flow rate:1 mL·min-1; column temperature:30℃; detection wavelength:245 nm; injection volume:20μL.
1.4 Analytical method of CYP2A6,2C19,2D6,2E1 activity of rat Liver microsomes in vitro
With coumarin, Mephenetoin, Dextromethorphan, chlorzoxazone being a cocktail probe, the levels of enzymic activity of CYP450 isoenzymes above were detected by LC-MS, which were suppressed by Wuji Pill with different compatibility in vitro.
LC-MS conditions of cocktail probe:
Clipeus-C8 column (30mm×2.1mm,5μm); mobile phase A:Water; mobile phase B:methanol; mobile phase C:5mM ammonium acetate; gradient elution program: Omin (90%A,10%B), 1.0min (90%A,10%B),2.0min (60%A,40%B),3.5min (60%A,40%B),3.51min (40%B,60%C),4.5min (60%B,40%C),4.51min (40% A,60%B),5.0min (20%A,80%B),6.0min (20%A,80%B),6.01min (90%A, 10%B),8.0min (90%A,10%B); running time:8min; flow rate:0.2mL·min-1; column temperature:25℃; injection volume:2μL; MS:ESI ion Source, Drying Gas Flow:10L·min-1; Drying Gas Tem:350℃; positive and negative mode while testing, MRM scan mode:7-hydroxycoumarin,163-189 positive ions; 4-hydroxy Meifen properly due,201-230 negative ions; the right of non-methane,269-233 positive ions; 6-hydroxy-chlorzoxazone 184-184 negative ions.
2 Analytical method of CYP450 activity of rat Liver microsomes in vivo
2.2 Analytical method of CYP1A2 activity of rat Liver microsomes in vivo
CYP1A2 substrate-phenacetin after oral administration in rats, HPLC determination of blood drug concentration in rats at different time points of phenacetin. The change of the pharmacokinetic parameters information may represent change of CYP450 activity.
HPLC conditions of Phenacetin, paracetamol as in vitro analysis.
2.2 Analytical method of CYP3A,2A6,2D6,2E1 activity of rat Liver microsomes in vivo
With testosterone, coumarin, Dextromethorphan, chlorzoxazone being a cocktail probe in vivo, the LC-MS conditions of cocktail probe:
Kromasil-C18 column (250mm×4.6mm,5μm); mobile phase A:methanol; mobile phase B:5mmol/L ammonium acetate; gradient elution program:Omin (28%A,72%B),5min (28%A,72%B),7min (50%A,50%B),20min (65%A, 35%B),30min (90%A,10%B); running time:35min; flow rate:1mL·min-1 (1:2 segregation after column, the actual flow rate of 1/3 flow); column temperature:35℃; injection volume:15μL. MS:API-ES ion source, in addition to 6-hydroxy-chlorzoxazone in negative scan mode, the remaining 7 tested substances (testosterone/6-hydroxy-testosterone, coumarin/7-hydroxy coumarin, dextrome-thorphan/right non-alkane and chlorzoxazone) are positive ion scan, drying gas velocity lOL/min, Nebulizer Pressure 40Psi, drying gas temperature of 350℃, capillary voltage 4000 is negative 4000, Fragmentor 70ev, testosterone positive ion scan-289.2, chlorineylsand were-187.1,-147.1 coumarin, dextromethorphan,-272.1,6-hydroxy-testosterone-305.2,6-hydroxy chlorzoxazone-184.1,7-hydroxyl-coumarin-163.1, right non-alky 1-258.1.
3 Statistics
Experimental data were statistical analysis by T value analysis of orthogonality, SPSS 11.5 and kinetica4.4 software.
RESULTS
1 Enzymic activity of rat liver microsomes
Rats after inducted by phenobarbital andβ-naphthoflavone, CYP450 enzyme concentration and activity in vitro can to meet the requirements of experiments. Determined by BCA kit microsomal protein content, protein concentration is 1.2g·L-1.
2 Effect of Wuji Pill on CYP1A2 activity of rat Liver microsomes in vitro
The IC50 of Rhizoma Coptidis, Fructus Evodiae Rutaecarpae, Radix Paeoniae Alba and 1-9# of different level Wuji Pill is:28.07,989.69,6633.28,57.92,104.38, 321.28,32.17,80.09,71.47,76.76,40.41 and 29.45μg(crude drug)/mL, respectively; Rhizoma Coptidis and 1-9 of Wuji Pill can suppress the enzymic activity of CYP1A2 significantly, and the capability of Rhizoma Coptidis in Wuji Pill of action on CYP1A2 can be modified by different composition of Fructus Evodiae Rutaecarpae and Radix Paeoniae Alba in Wuji Pill, and there are statistical difference among the IC50 of 1-9# of Wuji Pill; while the ratio of Rhizoma Coptidis and Fructus Evodiae Rutaecarpae raising up in Wuji Pill, Wuji Pill may supperess the enzymic of CYP1A2 strengthenly and with the ratio of Radix Paeoniae Alba raising up in Wuji Pill, the suppressed capability of Wuji Pill on CYP1A2 should be weaken oppositely.
3 Effect of Wuji Pill on CYP3A1/3A2 activity of rat Liver microsomes in vitro
The IC50 of Rhizoma Coptidis, Fructus Evodiae Rutaecarpae, Radix Paeoniae Alba and 1-9# of different level Wuji Pill is:38.96,871.96,15519.17,43.17,60.47, 276.12,133.40,118.08,88.47,64.36,35.13 and 39.91μg (crude drug)·mL-1, respectively; Rhizoma Coptidis and 1-9# of Wuji Pill can suppress the enzymic activity of CYP3A1/3A2 significantly, and the capability of Rhizoma Coptidis in Wuji Pill of action on CYP3A1/3A2 can be modified by different composition of Fructus Evodiae Rutaecarpae and Radix Paeoniae in Wuji Pill, and there are statistical difference among the IC50 of 1-9# of Wuji Pill; while the ratio of Rhizoma Coptidis raising up in Wuji Pill, Wuji Pill may supperess the enzymic activity of CYP1A2 strengthenly. Conclusion:The reason why Wuji Pill with different compatibility has different pharmacodynamics and pharmacokinetics characteristics is likely to lie in the difference of the capability of Wuji Pill with different compatibility on CYP3A1/3A2.
3 Effect of Wuji Pill on CYP2A6,2C19,2D6 and 2E1 activity of rat Liver microsomes in vitro
The IC50 of Rhizoma Coptidis, Fructus Evodiae Rutaecarpae, Radix Paeoniae Alba and 1-9# of different level Wuji Pill to CYP2A6 is:251.17,1925.82,2.18 e +09,735.93,380.84,1271.31,293.46,490.71,283.30,596.02,228.63,422.68μg (crude drug)·mL-1 respectively.
The IC50 of Rhizoma Coptidis, Fructus Evodiae Rutaecarpae, Radix Paeoniae Alba and 1-9# of different level Wuji Pill to CYP2C19 is:343.37,1713.23,20137.80, 339.31,240.80,245.52,136.59,192.45,277.41,174.95,128.18,133.19μg (crude drug)·mL-1 respectively.
The IC50 of Rhizoma Coptidis, Fructus Evodiae Rutaecarpae, Radix Paeoniae Alba and 1-9# of different level Wuji Pill to CYP2D6 is:145.77,1487.94,2.84 e +08,144.16,156.53,301.13,160.06,275.46,131.51,249.98,135.88,151.23μg (crude drug)·mL-1 respectively.
The IC50 of Rhizoma Coptidis, Fructus Evodiae Rutaecarpae, Radix Paeoniae Alba and 1-9# of different level Wuji Pill to CYP2E1 is:148.67,1461.11,8.02 e+31, 127.23,131.66,221.22,95.51,177.27,79.50,155.20,101.69,91.47μg (crude drug)·mL-1 respectively.
Rhizoma Coptidis and 1-9# of Wuji Pill can suppress the enzymic activity of CYP450 mentioned above significantly, and the capability of Rhizoma Coptidis in Wuji Pill of action on CYP450 can be modified by different composition of Fructus Evodiae Rutaecarpae and Radix Paeoniae in Wuji Pill, and there are statistical difference among the IC50 of 1-9# of Wuji Pill.
4 Effect of Wuji Pill on CYP1A2 activity of rat in vivo
The group covariance of Paracetamol concentration, and other multivariate analysis of variance show that Rhizoma Coptidis zero level (the control group or Fructus Evodiae Rutaecarpae, Radix Paeoniae Alba single administration group) compared with the high dose level of CYP1A2 enzyme activity was significant different. With the increase of Rhizoma Coptidis, activity tends to increase; and there are statistical difference among the IC50 of 1-9# of Wuji Pill.
5 Effect of Wuji Pill on CYP3A,2A6,2D6,2E1 activity of rat in vivo
Administration of Wuji Pill can significantly affected the process of coumarin in vivo in animals, further AUC, MRT parameters of univariate/multivariate analysis of variance, showed that effect of 3 factors of Wuji Pill (3 composed of herbs) on AUC of coumarin, Fructus Evodiae Rutaecarpae factor> Radix Paeoniae Alba factor> Rhizoma Coptidis factor.
univariate/multivariate analysis of variance of Dextromethorphan pharmaco-kinetic parameters show that:the control group, Fructus Evodiae Rutaecarpae group, Radix Paeoniae Alba group and 1# group was significantly reduced, effect of Wuji Pill on AUC of dextromethorphan, Rhizoma Coptidis factor> Radix Paeoniae Alba factor> Fructus Evodiae Rutaecarpae factor.
Different time points tested drugs concentration of 6-hydroxy-chlorzoxazone among experimental groups, the difference was significant, Fructus Evodiae Rutaecarpae zero dose and the middle dose level had significant differences; between middle dose and high dose level had significant difference.
Different time points tested drugs concentration of 7-hydroxycoumarin among experimental groups, the difference was significant, Fructus Evodiae Rutaecarpae zero dose and the middle dose level had significant differences; between middle dose and high dose level had significant difference.
Different time points tested drugs concentration of Dextrorphan among experimental groups, the difference was significant, Rhizoma Coptidis middle dose level compare with zero level and small dose level, the difference was significant; Fructus Evodiae Rutaecarpae small dose level compare with high dose level, the difference was significant; Radix Paeoniae Alba small dose level compare with middle dose level and high dose level, the difference was significant.
CONCLUSION
The reason why Wuji Pill with different compatibility has different pharmacodynamics and pharmacokinetics characteristics is likely to lie in the difference of the capability of Wuji Pill with different compatibility on CYP450.
引文
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