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fied by UPLC ESI Afatinib Q TOF MS and 1H NMR. 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. The UPLC system developed for emodin had a run time of 4 min plus a linear calibration curve over the concentration range of 0.6125 40 M . The intra and inter day variabilities at 1.25, 10, and 40 M of emodin were much less than 4.2 and 3.8 , respectively. In microsomal incubation samples, a single new peak eluted at 1.92 min . A UPLC ESI Q TOF MS running at a negative ion mode was utilised to determine the MS spectrum with the metabolite. The mass spectra of this metabolite exhibited a molecular ion at m z 445.0780, calculated as C21H17O11: 445.
0776, Afatinib which corresponded to the molecular weight of emodin glucuronide, as well as the main fragment ion at m z 269.0462, which corresponded to the molecular weight of emodin . LC MS MS study also indicated that all metabolites generated from a variety of microsomes of diverse species showed identical mono glucuronide of emodin . The UV spectra of emodin glucuronide and emodin were similar, which were supportive with the notion that the new eluted peak is closely related to emodin. 1H NMR spectra with the metabolite displayed quite similar signals with those of emodin except for the signals derived from an extra sugar moiety which was determined to be glucuronide group from its H 1 signal at 5.14 and H 5 signal at 4.21 . The location of glucuronide group was confirmed to be at 3 OH by the observation of NOE correlations between the anomeric proton with both H 4 and H 2 within the NOESY spectrum shown in Fig.
1d. Depending on the above evidences, the metabolite was identified as emodin 3 O D glucuronide . Because exactly the same glucuronide was identified in all glucuronidation reactions working with liver microsomes of any species or gender, emodin Everolimus 3 O D glucuronide was the only glucuronide formed within the present study. Glucuronidation of Emodin by Rat Liver Microsomes Emodin was rapidly glucuronidated by rat liver microsomes . Following 15 min, only 20 of emodin was left . Following incubation occasions of 30 min, 1 h, and 2 h, percent remaining were 9.73 , 5.73 , and 1.87 , respectively. Phase I Metabolism of Emodin by Rat Liver Microsomes For phase I oxidation reaction performed working with identical concentration of rat liver microsomes, the percent emodin remaining was 84.
81 right after 15 min of reaction time. Following reaction occasions of 0.5, 1, and 2 h, the percent remaining were 65.53 , 42.53 , and 28.35 , respectively . For that reason, it was clear that oxidative metabolism was a minimum of five occasions slower HSP than glucuronidation. In oxidative metabolism, a single principal metabolite was identified, which was eluted at the retention time of 2.07 min plus a molecular ion at 285.16 Da, 16 more than that of emodin , indicating that the compound can be a hydroxylated metabolite of emodin . The MS MS spectrum of item ion at m z 255 and m z 268 suggested that the metabolite should be hydroxyemodin, as reported previously . The MS2 profile with the hydroxyemodin is noticed in Fig. 2a, but we were unable to assign the position with the hydroxylation.
Metabolism of Emodin in a Mixed Oxidation and Glucuronidation Reaction System The mixed system of oxidation and glucuronidation reaction was utilised to determine Everolimus the key pathway of metabolism of emodin by using male rat liver Afatinib microsomes at 1.67 mg mL with both oxidation and glucuronidation reaction cofactors. Detectable amount of emodin glucuronide was observed within 6 min of incubation, and emodin was metabolized nearly totally within 1 h. The metabolite was confirmed to be emodin 3 O D glucuronide by LCMS MS, which was the only metabolite identified within the mixed reaction system. There were no detectable amounts of hydroxyemodin identified within the mixed reaction system, confirming earlier observation that glucuronidation reaction was significantly far more rapid than oxidation reaction.
Intestinal Absorption and Metabolism of Emodin Absorption of emodin displayed regional difference in male but not in female rats . On the other Everolimus hand, excretion of emodin glucuronide displayed region dependence in both male and female rats . The amounts of emodin glucuronide excreted in duodenum were significant higher than that in jejunum, followed by ileum and colon in male rats . In female rats, the rank order of amounts of metabolite excreted was jejunum≈duodenum ileum colon . The amounts of emodin absorbed in each and every with the four regions of female rat intestine were higher than that within the male rats , and range of the increase was 27 44 . In contrast, amounts of emodin glucuronide excreted were higher in each and every with the four segments of intestine within the male rats than the female rats , as well as the range of the increase was 40 67 , indicating somewhat larger difference in metabolism than in excretion. Concentration Dependent Glucuronidation of Emodin by Rat Intestinal Microsomes To determine when the above observed pattern of metabolite excr