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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 method developed for emodin had a run time of 4 min and a linear calibration curve over the concentration selection 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 damaging ion mode was applied to decide the MS spectrum in 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, and the major 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 different species showed identical mono glucuronide of emodin . The UV spectra of emodin glucuronide and emodin were similar, which were supportive in the notion that the new eluted peak is closely related to emodin. 1H NMR spectra in the metabolite displayed quite similar signals with those of emodin except for the signals derived from an additional 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 . Given that exactly the same glucuronide was identified in all glucuronidation reactions making use of 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 quickly glucuronidated by rat liver microsomes . Soon after 15 min, only 20 of emodin was left . Soon after incubation times 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 making use of identical concentration of rat liver microsomes, the percent emodin remaining was 84.
81 immediately after 15 min of reaction time. Soon after reaction times 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 at least five times slower HSP than glucuronidation. In oxidative metabolism, a single primary metabolite was identified, which was eluted at the retention time of 2.07 min and a molecular ion at 285.16 Da, 16 more than that of emodin , indicating that the compound is really a hydroxylated metabolite of emodin . The MS MS spectrum of product ion at m z 255 and m z 268 suggested that the metabolite should be hydroxyemodin, as reported previously . The MS2 profile in the hydroxyemodin is noticed in Fig. 2a, but we were unable to assign the position in the hydroxylation.
Metabolism of Emodin inside a Mixed Oxidation and Glucuronidation Reaction System The mixed system of oxidation and glucuronidation reaction was applied to decide Everolimus the main 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 completely 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 more fast 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 substantial 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 in the four regions of female rat intestine were higher than that within the male rats , and selection of the increase was 27 44 . In contrast, amounts of emodin glucuronide excreted were higher in each and every in the four segments of intestine within the male rats than the female rats , and the selection 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 decide when the above observed pattern of metabolite excr