Metabolism studies on hydroxygenkwanin and genkwanin in human liver microsomes by UHPLC-Q-TOF-MS

Abstract

Hydroxygenkwanin (HYGN) and genkwanin (GN) are major constituents of Genkwa Flos for the treatment of edema, ascites, cough, asthma and cancer. This is a report about the investigation of the metabolic fate of HYGN and GN in human liver microsomes and the recombinant UDP-glucuronosyltransferase (UGT) enzymes by using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). An on-line data acquisition method multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS) was developed to trace all probable metabolites. Based on this analytical strategy, three phase I metabolites and seven glucuronide conjugation metabolites of HYGN, seven phase I metabolites and 12 glucuronide conjugation metabolites of GN were identified in the incubation samples of human liver microsomes. The results indicated that demethylation, hydroxylation and o-glucuronidation were main metabolic pathways of HYGN and GN. The specific UGT enzymes responsible for HYGN and GN glucuronidation metabolites were identified using recombinant UGT enzymes. The results indicated that UGT1A1, UGT1A3, UGT1A9, UGT1A10 and UGT2B7 might play major roles in the glucuronidation reactions. Overall, this study may be useful for the investigation of metabolic mechanism of HYGN and GN, and it can provide reference and evidence for further experiments.

Identification of Metabolites of Eupatorin in Vivo and in Vitro Based on UHPLC-Q-TOF-MS/MS

“…With regard to the isomers of metabolites, Clog P values calculated by ChemDraw 14.0 were used to further distinguish them. Generally speaking, the larger the Clog P value, the longer the retention time will be in the reversed-phase chromatography system [25,26,27].…”

A Complete Study of Farrerol Metabolites Produced In Vivo and In Vitro

“…The corresponding ESI-MS 2 spectrum featured product ions with m/z 281.0782, 255.0997, and 253.0843 produced by loss of H 2 O, CO 2 , and H 2 O + CO, respectively, while the product ion with m/z 205.0483 was formed through C2–C1′ bond cleavage. Additionally, secondary ions with m/z 179.0333 and 119.0493 were produced by retro-Diels−Alder (RDA) cleavage of ring C, and this reaction was concluded to be a representative flavonoid fragmentation pathway allowing rapid metabolite identification [21,27,28]. The MS/MS spectra and fragmentation pathways of farrerol are presented in Figure 3.…”

“…Moreover, UGTs primarily participate in the glucuronidation of flavanones and flavones. UGT1A1 and UGT1A9 have unique characteristics in catalyzing the glucuronidation at 5-position of flavones, and UGT1A9 and UGT1A10 are responsible for producing the glucuronidation metabolites with hydroxyl groups at the B ring [27]. UGT1A1 and UGT1A9 could involve in formation of metabolites M19, M25, M26 and M28.…”

“…Drug metabolism is important for the study of drug safety and toxicity, and therefore plays a vital role in drug discovery and clinical use [18]. The use of rats for in vivo metabolism investigations has been proven to be feasible and efficient [24,25,26], while liver microsomes are well suited for metabolite identification, metabolite preparation, and toxicity research in vitro [19,27]. Herein, 42 farrerol in vivo metabolites and 15 farrerol in vitro metabolites were identified.…”

“…A classic incubation mixture (total volume = 200 μL) comprising 50 μmol/L farrerol, 0.8 mg/mL microsomal protein, 2.7 mmol/L MgCl 2 , and 20 μg/mL alamethicin was used for the cascade reaction [27]. As in the case above, incubation was carried out in 10× PBS buffer, and pre-incubation was performed for 5 min at 37 °C in an incubator shaker.…”