| Osteoporosis(OP) had become one of the common diseases of postmenopausal women and old men. The incidence of OP were about 20% in men and 50% in postmenopausal women. The therapeutic drug of OP can be classed into two groups. One group was the inhibitor of bone absorbance, the other group was the accelerant of bone formation. The inhibitor of bone absorbance was predominantly used for the therapy of OP. Ipriflavone(IP) was a promising drug belonging to inhibitor of bone absorbance. IP was one of the synthetic isoflavones. As a drug in common use for old people, IP was often administered with other drugs such as propranolol, estradiol. It had been observed that many drugs may occur interaction resulting in inefficacy due to the inhibition or induction of metabolic enzymes in liver. In this study, the determination of IP in rat microsomes by RP-HPLC was established and the metabolism of IP in rat microsomes was investigated as well as the interaction of IP and other drugs.1.Development of analytical method for determination of ipriflavone in ratliver microsomes by RP-HPLC Chromatographic ConditionsChromatographic column: Nova-park C18(20cm X 4.6mm, 4 u m); mobilephase:acetonitrile-0.1%aceticacid(60:40,v/v);flow-rate:1.0 ml · min-1; wavelength of UV detector: 250nm.Experiment procedureThe incubation mixture containing microsomal protein was bubbled in oxygen for 1 min. IP was added as substrate. NADP/NADPH was added to start enzymatic reaction after preincubation for 5 min at 37 C. At designed time, Chloroform was added and mixed. DL111-IT was added as internal standard. The incubation mixture was extracted for 1 min and centrifuged. 0.5ml of the organic layer was evaporated to dryness. The residue was dissolved with 100 ul mobile phase. A 20ul aliquot was analyzed by HPLC. ResultsThe standard curve for IP in spiked microsomal incubate was linear from 1 ~ 100 u g · ml-1. The regression equations of the standard curve based on the concentration of IP versus the ratio of the peak-area of IP to IS was Y=0.06263X-4.8215 X 10'3(r=0.9998). The limit of detection was 0.1 u g · ml-1 (S/N>3) and the limit of quantification was 0.71 ?.03p.g ·ml-1(RSD=4.8%, n=3). The relative standard deviation for intra-assay in low,middle,high concentration were 3.6%, 3.5%, 3.0%, respectively. The relative standard deviation for inter-assay in low,middle,high concentration were 8.0%, 5.0% 4.4%, respectively. The mehod afforded recoveries in low,middle,high concentration were 110.2%, 104.1%, 96.9%, respectively.2.Metabolism of ipriflavone in rat liver microsomes pretreated with different inducers Experiment procedureThe microsomes were pretreated with p-naphthoflavone (BNF), dexamethasone (DEX), Phenobarbital (PB), diphenytriazol(DLlll-IT), respectively. The incubation mixture was bubbled in oxygen for 1 min. IP was added as substrate. NADP/NADPH was added to start enzymatic reaction after preincubation for 5 min at 37 C. The incubation of substrate concentration-incubation time curve lasted 3 min, 10 min, 20 min, respectively. The incubation of determination of enzyme kinetic parameter lasted 3 min. Chloroform was added and mixed. DL111-IT was added as internal standard. The incubationmixture was extracted for 1 min and centrifuged. 0.5ml of the organic layer was extracted and evaporated to dryness. The residue was dissolved with 100u] mobile phase. A 20ul aliquot was analyzed by HPLC. ResultsThe metabolism of IP in PB group showed no significant difference (P>0.05) comparing with control group after incubation for 20 min, while DEX group, BNF group, DL111-IT group showed significant differences(P<0.05, P<0.01, P<0.001, respectively). Peak area of the response of main metabolism (Ml) of IP in microsomes pretreated with PB, DEX, BNF, DL111-IT were larger than those in microsome untreated at any time. Peak area of the response of main metabolism (Ml)of IP in DEX group was same as those in BNF group, which were larger than those in PB group. Peak area of the response of main metabolism (M2) of IP in PB group and DEX group were a little larger than...
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