| AIMWuji 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.METHODS1 Analytical method of CYP450 activity of rat Liver microsomes in vitroAdministration 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 microsomesWistar 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 vitroWith 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 vitroWith 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 vivo2.2 Analytical method of CYP1A2 activity of rat Liver microsomes in vivoCYP1A2 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 vivoWith 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 StatisticsExperimental data were statistical analysis by T value analysis of orthogonality, SPSS 11.5 and kinetica4.4 software.RESULTS1 Enzymic activity of rat liver microsomesRats 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 vitroThe 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 vitroThe 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 vitroThe 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 vivoThe 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 vivoAdministration 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.CONCLUSIONThe 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.
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