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lly exactly the same as those published previously Doxorubicin . Briefly, they were as follows: Microsomes , magnesium chloride , saccharolactone , alamethicin , various 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 regular. Afterwards, the samples were centrifuged at 13,000 rpm for 15 min along with the supernatant employed for injection. To control the extent of metabolism to 30 parent compound, various combinations of microsomal protein amounts and incubation time were tested in preliminary studies, and 10 min was found to be the ideal 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 exactly the same as the published procedures Imatinib . Briefly, the procedures were as follows: Microsomes was mixed with remedy A and remedy B inside a 50 mM potassium phosphate buffer . The mixture was preincubated at 37 C for 5 min, and emodin stock remedy was then added. The final mixture was incubated for a predetermined time period at 37 C, along with the reaction was stopped by the addition of 50 L of 94 acetonitrile 6 glacial acetic acid containing 50 M testosterone as the internal regular.
CH2Cl2 was then added towards the final remedy, vortexed for 30 s, and centrifuged at 3,500 rpm for 15 min. Right 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 no NADPH producing method served as the control. All reactions were performed at least three times in three duplicates. Simultaneous Phase I and Glucuronidation of Emodin Considering that emodin may undergo phase I oxidation and glucuronidation simultaneously, Imatinib a mixed method of oxidation and glucuronidation reaction was employed to establish the primary 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 create exactly the same results. Determination of Molar Extinction Coefficients of Emodin Glucuronide Due to the lack of emodin glucuronide standards, an emodin regular curve was employed for quantitation of emodin glucuronide by using a conversion aspect , as was done previously in our lab for isoflavones . The conversion aspect, 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 eliminate emodin.
The extracted aqueous sample was subsequently divided into two equal parts; 1 element was incubated with water and then analyzed by UPLC along with the other 1 by hydrolysis with glucuronidase at 37 C for Imatinib 30 min and then analyzed by UPLC. The difference in peak places of metabolite and emodin obtained from the samples just before and soon after the hydrolysis, which were represented as Peak areaM and Peak areaE, was calculated to be the ratio K ? Peak areaM Peak areaE e T . Therefore, the concentration of metabolite could be estimated using emodin regular curve. The average SD conversion aspect was 1.0054 0.023 at a wavelength of 254 nm, determined separately at three various concentrations . UPLC and LC MS MS Analysis of Emodin and its Glucuronides The circumstances 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 items in aqueous remedy was extracted with dichloromethane three times. The aqueous fraction was loaded onto an ODS column and washed using pure water. The mono glucuronide emodin was eluted using a solvent of H2O MeOH . The structure of mono glucuronide emodin was identi