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lly the same as those published previously Doxorubicin . Briefly, they were as follows: Microsomes , magnesium chloride , saccharolactone , alamethicin , diverse concentrations of substrate inside a 50 mM potassium phosphate buffer , and UDPGA were mixed. The mixture was incubated at 37 C for a predetermined time period . The reaction was stopped by the addition of 100 L of 94 acetonitrile 6 glacial acetic acid containing 50 M testosterone as the internal normal. Afterwards, the samples were centrifuged at 13,000 rpm for 15 min and the supernatant employed for injection. To manage the extent of metabolism to 30 parent compound, diverse combinations of microsomal protein amounts and incubation time were tested in preliminary studies, and 10 min was identified to be the best incubation time when we employed a microsomal protein concentration of 0.
026 mg mL at emodin concentrations of 30 40 M, 0.013 mg mL at emodin concentrations of Doxorubicin 10 20 M, and 0.005 mg mL at emodin concentrations at or beneath 7.5 M, respectively. Phase I Metabolism of Emodin The procedure for conducting phase I reaction was essentially the same as the published procedures Imatinib . Briefly, the procedures were as follows: Microsomes was mixed with solution A and solution B inside a 50 mM potassium phosphate buffer . The mixture was preincubated at 37 C for 5 min, and emodin stock solution was then added. The final mixture was incubated for a predetermined time period at 37 C, and the reaction was stopped by the addition of 50 L of 94 acetonitrile 6 glacial acetic acid containing 50 M testosterone as the internal normal.
CH2Cl2 was then added towards the final solution, vortexed for 30 s, and centrifuged at 3,500 rpm for 15 min. Soon after the aqueous and protein layers were aspirated out, the CH2Cl2 layer was transferred to a clean tube and dried under nitrogen gas. The residues were dissolved NSCLC in 110 L of water and methanol and injected into UPLC for analysis. Reaction samples with out NADPH generating method served as the manage. All reactions were performed a minimum of three times in three duplicates. Simultaneous Phase I and Glucuronidation of Emodin Since emodin could undergo phase I oxidation and glucuronidation simultaneously, Imatinib a mixed method of oxidation and glucuronidation reaction was employed to decide the key pathway of metabolism of emodin in vitro.
The procedures basically combined what was described earlier for separate oxidative and glucuronidated reactions, Doxorubicin and all compounds added previously for those reactions were added for the mixed reaction as well, and as a result, both reaction systems were expected to generate the same results. Determination of Molar Extinction Coefficients of Emodin Glucuronide Because of the lack of emodin glucuronide standards, an emodin normal curve was employed for quantitation of emodin glucuronide by using a conversion factor , as was accomplished previously in our lab for isoflavones . The conversion factor, which is the ratio between the molar extinction coefficient of emodin glucuronide and emodin, was determined by the following procedures: An aqueous sample containing emodin glucuronide and emodin was extracted three times with dichloromethane to get rid of emodin.
The extracted aqueous sample was subsequently divided into two equal parts; 1 portion was incubated with water and then analyzed by UPLC and the other 1 by hydrolysis with glucuronidase at 37 C for Imatinib 30 min and then analyzed by UPLC. The difference in peak locations of metabolite and emodin obtained from the samples before and following the hydrolysis, which were represented as Peak areaM and Peak areaE, was calculated to be the ratio K ? Peak areaM Peak areaE e T . Consequently, the concentration of metabolite could be estimated making use of emodin normal curve. The average SD conversion factor was 1.0054 0.023 at a wavelength of 254 nm, determined separately at three diverse concentrations . UPLC and LC MS MS Analysis of Emodin and its Glucuronides The conditions employed to analyze emodin and its metabolites were as follows: method, Waters Acquity? UPLC with photodiode array detector and Empower software program; column, BEH C18, 1.
7 m, 2.1 50 mm; mobile phase B, 100 acetonitrile, mobile phase A, 100 aqueous buffer ; flow rate, 0.4 mL min; gradient, 0 to 0.1 min, 85 A, 0.1 to 1.8 min, 85 60 A, 1.8 to 2.2 min, 60 40 A, 2.2 to 2.8 min, 40 85 A, 2.8 to 3.2 min, 85 A, wavelength, 254 nm for emodin and its glucuronide and testosterone; and injection volume, 10 L. The Imatinib test linear response range was 0.625 100 M for emodin. The mass spectrometer parameters were set as follows: capillary voltage, 4.5KV; ion source temperature, 350 C, desolvation temperature, 108 C; nebulizer gas , nitrogen, 40 psi; turbo gas , argon gas, 20 psi. A mixture of reaction merchandise in aqueous solution was extracted with dichloromethane three times. The aqueous fraction was loaded onto an ODS column and washed making use of pure water. The mono glucuronide emodin was eluted making use of a solvent of H2O MeOH . The structure of mono glucuronide emodin was identi