| OBJECTIVESTo research the effects of glycyrrhizic acid on metabolism kinetics for toxic dosage brucine and investigate a new antidotal pathway for brucine-induced toxicity:enhance poison excretion by mdrl up-regulation and P-gp induction.To study the disposition kinetics and brain/plasma concentration ratio of brucine after a single and multiple dose in mice which pre-treated with glycyrrhizic acid and verapamil; to analyze and compare the expression of mdr1a in brain tissues from different groups in order to disscuss the mechanism of detoxication.METHODS1. Dose and sample collection1.1144 male KM mice (30±3 g) were equally divided into three groups by completely random design. Each group was injected intraperitoneally with normal sodium (B1), glycyrrhizic acid (40 mg·kg-1) (G) and verapamil (5 mg·kg-1) (V) for 7 days. In the 8th day, every mouse was injected intraperitoneally with brucine(30 mg·kg-1). Blood and brain samples were collected at 5,15,30,60,120,240,420 and 600 min. All samples were stored in-70℃.1.2 144 male KM mice (30±3 g) were equally divided into three groups by completely random design. Each group was injected intraperitoneally with brucine (30 mg·kg-1) (B7), glycyrrhizic acid (40 mg·kg-1) add brucine (30 mg·kg-1) (B+G), and verapamil (5 mg·kg-1) add brucine (30 mg·kg-1) (B+V) for 7 days. Blood and brain samples were collected at 5, 15,30,60,120,240,420 and 600 min.1.3 15 male KM mice (30±3 g) were equally divided into five groups by completely random design. Mice in control group were injected intraperitoneally with normal sodium for 7days. Pre-treatment for the groups of Gb,Vb,B7b,B+Gb were the same as G,V,B7,B+G. Brain samples were collected at 2h after the last injection and stored in liquid nitrogen.2. Sample analysis2.1 The concentrations of brucine in blood and brain samples were determined by UPLC-MS/MS.2.2 Real-time fluorescence quantitative RT-PCR was used to determine the expression of mdr1a in brain tissues of part 1.3. RESULTS 1. Validation of sample analysis methodIn blood, linear range was 5.016～5016 ng·mL-1 for brucine; In brain, linear range was 1.003～241.8 ng·mL-1. Recovery was 91%～113%. The inter-day and intra-day precision(RSD) were less than 11.2%.2. Metabolism kinetics and brain/plasma concentration ratio after intraperitoneally injection of brucine2.1 AUC0→10h in blood(μg·min·mL-1) was 226.13±22.11,255.76±32.40, 209.99±12.36,254.22±39.81,355.90±32.64 and 248.79±40.07 for B1,G,V,B7,B+G and B+V, respectively. There had great difference between group G and group B1, group B+G and group B7(P<0.05).2.2 AUC0→10h in brain(μg·min·mL-1) was 13.69±0.91,15.08±5.56, 17.89±1.24,9.43±2.32,12.57±1.10 and 12.53±1.29 for B1,G,V,B7,B+G and B+V, respectively. There had great difference between group V and group B1, group B+G,B+V and group B7(P<0.05).2.3 AUC0→10h of brain/plasma concentration ratio (ng·h·mL-1) was 1.57±0.46,0.98±0.13,2.08±0.33,1.03±0.31,0.48±0.32 and 1.50±0.66 for B1, G, V, B7, B+G and B+V, respectively. There had great difference between group G,V,B7 and group B1; group B+G and group B7 also had great difference(P<0.05).3. The expression of mdr1a mRNA in brain tissues between different groups In group G and B+G, the expression of mdr1a mRNA increased in brain tissues compared to mice injected with normal sodium.CONCLUSIONS1. Continue pre-treatment with glycyrrhizic acid will not accelerate the elimination of brucine from blood, but will accelerate the elimination from brain. The experiment prove that glycyrrhizic acid may up-regulate the P-gp's function in mice brain. Verapamil could reverse the decreased trend of drug disposition in brain, so brucine may be the substrate of P-gp in blood-brain barrier.2. Brucine may have self-induction effect. Its influence on brain concentration is greater than blood. The result shows that brucine may has more significant effects on transporters.3. The RT-PCR results reveal that continue treated with glycyrrhizic acid could induce the expression of mdr1a mRNA in mice brain. So these drugs could decrease the distribution in brain through inducing P-gp's expression. The assumption that through up-regulating mdrl and inducing the expression of P-gp to accelerate the elimination of brucine from brain is reasonable and feasible. |
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