Evaluation Of Total Flavonoids Of Bidens Bipinnata L On Cytochrome P450 Activity By Cocktail Probe Drugs

Objective:Bidens bipinnata L., a well-known, traditional Chinese medicine, has been used to treat hepatitis in clinics for many years, and also can be used in the treatment of Malaria,rheumatism et al. Some satisfactory pharmacological activities have been found in anti-inflammatory, antioxidation, anti-fibrosis, etc. As the main active part of Bidens bipinnata L., total flavonoids of Bidens bipinnata L.(TFB) has a broad prospect in clinical application. The use of herbal medication or its active constituent has been on the rise as an alternative or complementary therapy worldwide. Despite the potential herb-drug interactions for pharmacokinetic and pharmacodynamic, many people take herbal medications in combination with prescribed Western medication. In recent years, a dramatic increase in the number of herb-drug interactions has been reported and have aroused attention with regard to clinic drug safety. Human hepatic cytoehrome P450(CYP450) enzymes play a dominant role in the metabolism of drugs. To evaluate the drug-herb interactions between TFB and other drugs, it is important to investigate the activity alteration after the use of TFB. The purpose of this study is to find whether TFB influences the effect on rat cytochrome P450(CYP450) enzymes by using cocktail probe drugs in vivo. In this way, a guide of TFB use can be providing for clinical treatment in future.Methods:We developed a High performance liquid chromatography(HPLC) method to detect the Cocktail approach which reflect the metabolic activities of different CYP isoforms. Then we used it to evaluate the effects of TFB on the activities of CYP isoforms CYP2E1, CYP2C9 and CYP3A4 in vivo. Cocktail probe solution was prepared by mixing chlorzoxazone, tolbutamide and midazolam, which are the specific probe drugs of CYP450 enzyme isoforms, represented the activity of CYP2E1, CYP2C9 and CYP3A4 respectively. Rats were divided into five groups randomly: blank control group(NS), normal dose test group of TFB(N-TFB), high dose test group of TFB(H-TFBT), inhibition group, induction group. TFB was administered by oral for 7 days at a dose of 80mg·kg-1 body weight for N-TFB group and at a dose of 240 mg·kg-1 for H-TFB group. NS was administered for blank control group with the same volume as N-TFB group or H-TFB group. Inhibition group and induction group were administered Cimetidine and Phenobarbital at a dose of 50 mg·kg-1 body weight respectively by intraperitoneal injection for 7 days. On the 8th day, all groups accepted Cocktail solution by injecting caudal vein. Blood samples of each rat were collected pre-dose(0 h) and at a series of time-points after probe drugs administration through the eyeball vein. The concentration of every probe drug at the series time in plasma detected by HPLC, the Pharmacokinetic Parameters was calculated by WinNolin 2.1. The effects of TFB on the activities of CYP2E1, CYP2C9 and CYP3A4, which were the three important isoforms of CYP450, were evaluated indirectly by comparing the Pharmacokinetic Parameters of the test group with the other groups.Results:1. We established a High performance liquid chromatography method which could simultaneously determine the concentrations of chlorzoxazone, tolbutamide, and midazolam in plasma. The detection limits range from 0.1-50μg·ml-1 for chlorzoxazone, tolbutamide and midazolam. The intra-day and inter-day precisions for three probe substrates were 4.22%-6.17% and 4.38-11.84% respectively, the accuracy of three probe substrates ranged from 92.71%-109.79%. The limit of quantification(LOQ) was 0.1μg·ml-1 for chlorzoxazone, tolbutamide and midazolam. The precision, accuracy and sensitivity were suitable for analyzing the three probe substrates in plasma.2. Through comparing the pharmacokinetic parameters of high dose test group of TFB with NS groups, we found that AUC0-t, AUC0-∞ of chlorzoxazone significantly increased, and CL significantly decreased(P<0.05), MRT0-∞ and T1/2 were longer than NS group but no statistic difference; The variation of pharmacokinetic parameters was similar as cimetidine group(P>0.05). AUC0-∞, MRT0-∞ and T1/2 of tolbutamide significantly decreased, and CL was significantly increased(P<0.05); AUC0-t of midazolam was significantly increased(P<0.05),T1/2、AUC0-∞、MRT0-∞ were increased and CL was decreased but no significant difference(P>0.05).3. We Compared the pharmacokinetic parameters of normal dose test group of TFB with blank control groups, AUC, MRT and T1/2 of chlorzoxazone increased, and CL decreased, but there were no statistically significant differences from NS group; MRT0-∞ and T1/2 of tolbutamide significantly increased(P<0.05), The variation of pharmacokinetic parameters was similar as cimetidine group(P>0.05); Similar variations were founded in the pharmacokinetic parameters of midazolam, but there was no significant difference.Conclusions:1. The present method provides an effective, fast analytical tool for the three-probe drug cocktail. Finally, the method was suitable for determining the plasma concentration of these compounds and evaluating the CYP2E1, 2C9 and 3A4 activities in vivo interaction studies.2. Treatment with a high dose of TFB had inhibitory effects on rat CYP450 enzymes, The inhibition of TFB on CYP2E1, CYP2C9 enzyme activities at a high dose was similar to cimetidine. Treatment with TFB at a normal dose inhibited the activity of CYP2C9, no significantly inhibitory effect on CYP2E1 and CYP3A4 enzymes was founded, but we also need take attention of the potential herb-drug interaction while the co-administered drugs were metabolized by CYP2E1, CYP2C9 or CYP3A4.