ABSTRACT
@#To investigate the influential mechanism of total flavonoids from Abelmoschus Manihot (HKZ) on cytochrome P450 (CYP450) isoforms in human liver microsomes and to verify its effect on the most significantly inhibited subtype CYP2C9 in rats.The inhibitory effects of HKZ on human CYP3A4, CYP2C9, CYP2C19, CYP2E1, CYP1A2 and CYP2D6 were evaluated through the cocktail method using ultra-performance liquid chromatography tandem mass spectrometry, then its inhibitory mechanism was investigated and kinetic parameters of enzyme inhibition were calculated By comparing the pharmacokinetic behaviors of tolbutamide after single or multiple administration of 200 mg/kg HKZ and equal dose of CMC-Na in rats, the effects of HKZ on CYP2C11 enzyme (CYP2C9 isoenzyme) was estimated.The results indicated the significant inhibitory effect of HKZ on CYP2C9 and CYP2E1 with IC50 of 3.22 and 8.64 μg/mL, respectively. Also, it showed certain inhibitory ability on other isoforms with IC50 of 20-30 μg/mL.As demonstrated, HKZ may not be a time-dependent inhibitor which competitively inhibited CYP2E1 and CYP2C9 with Ki of 3.84 and 6.33 μg/mL.In contrast, it showed noncompetitive inhibition on CYP3A4 mediated testosterone-6β-hydroxylation and midazolam-4-hydroxylation reaction with Ki of 7.37 and 3.32 μg/mL.It was also a noncompetitive inhibitor of CYP1A2, CYP2D6 and CYPC219 with Ki values of 8.66, 11.49 and 21.94 μg/mL. HKZ did not change the pharmacokinetic parameters of CYP2C11 probe substrate tolbutamide in rat, but it affected the AUC0-t, cmax of 4-hydroxytolubutamide (P < 0.05). Therefore, drug-drug interaction mediated by CYP450 should be considered in clinical study.
ABSTRACT
OBJECTIVE： To establish a method for simultaneous determination of 9 kinds of organic solvents residues in total flavonoids extracts from Abelmoschus manihot. METHODS： Headspace GC was adopted to determine the contents of 9 kinds of organic solvents residues in total flavonoids extracts from A. manihot， such as benzene， acrylonitrile， methyl methacrylate， toluene， 1，2-dichloroethane， xylene， chlorobenzene， styrene and divinylbenzene. The determination was performed on Agilent DB-WAX capillary column （30 m×0.25 mm， 0.25 μm） through temperature-programmed route. The inlet temperature and FID detector temperature were set at 250 ℃. The carrier gas was nitrogen with the flow rate of 1.2 mL/min. The split ratio was 10 ∶ 1. The containers of headspace injector were in equilibrium at 90 ℃ for 45 min and the sample size was 1 mL. RESULTS： The separation degree among the peaks of 9 components was greater than 1.5， and the blank solvent （10％ N-methyl pyrrolidone aqueous solution） had no interference. The linear ranges of them were 0.16-1.21， 0.80-6.03， 1.61-12.09，1.62-12.12， 0.16-1.21， 1.60-12.01， 0.81-6.11， 1.60-12.03， 0.80-6.03 μg/mL， respectively （r≥0.999 4）. The limits of quantitation were 0.162 08， 0.201 08， 0.080 6， 0.080 768， 0.161 92， 0.400 36， 0.040 712， 0.026 736， 0.013 395 μg/mL； the limits of detection were 0.040 52， 0.040 216， 0.026 87， 0.026 9，0.040 48， 0.080 072， 0.013 57， 0.008 912， 0.004 465 μg/mL， respectively. RSDs of precision （n＝5） and reproducibility （n＝6） tests were all lower than 5％. Average recoveries were 99.41％-111.27％（RSD＜9％， n＝9）. Above 9 residual solvents were not detected in 3 batches of total flavonoids extracts from A. manihot. CONCLUSIONS： Established method is simple， accurate and reliable， and can be applied for simultaneous detection of 9 kinds of organic solvents residues in total flavonoids extracts from A. manihot.
