title: Comparative metabolism of tussilagone in rat and human liver microsomes using ultra-high-performance liquid chromatography coupled with high-resolution LTQ-Orbitrap mass spectrometry.
abstract: BACKGROUND
  Tussilagone is a major component in Tussilago farfara that has been widely used as an anti-tussive herbal medicine for the treatment of bronchitis, cough and asthmatic disorders in the clinic. However, its metabolism has been poorly investigated. In order to clarify its in vitro metabolism, a comparative analysis of its metabolic profile in rat liver microsomes (RLMs) and human liver microsomes (HLMs) was carried out. Further, the cytochrome P450 isoforms (CYPs) involved in the metabolism were investigated.
  
  
  METHODS
  In this work, the biotransformation of tussilagone in RLMs and HLMs was compared using ultra-high-performance liquid chromatography coupled with high-resolution LTQ-Orbitrap mass spectrometry (UHPLC/HRMS) and the CYPs involved in the metabolism were further investigated by recombinant human CYP enzymes.
  
  
  RESULTS
  Totally, nine metabolites of tussilagone were identified in RLMs and HLMs based on the proposed MS/MS fragmentation pathways of tussilagone and the accurate MS/MS spectra. Among them, one metabolite (M9) was detected in both RLMs and HLMs while the other eight metabolites were only detected in HLMs. Three hydroxylation metabolites (M6, M7 and M8) were detected in the assay with individual recombinant P450s incubation. M6 was detected in all CYPs except CYP2A6 while M7 and M8 were only observed in CYP3A4.
  
  
  CONCLUSIONS
  The HR-ESI-MS/MS fragmentation behavior of tussilagone and its metabolic profile in RLMs and HLMs were investigated for the first time. The results demonstrated that the biotransformation of tussilagone involved hydrolysis of ester bonds at C-14 and hydroxylation in the side chains at C-12, C-5' or C-6'. Among the CYPs, CYP3A4 played an important role in the hydroxylation reaction of tussilagone in vitro. Furthermore, the results indicated a species-related difference in the metabolism of tussilagone between RLMs and HLMs. This work provided basic information for the metabolism of tussilagone in RLMs and HLMs, which would help to better understand the pharmacological activities of tussilagone.
authors.affiliation: Department of Pharmacy, Hebei North University, Zhangjiakou, 075000, China.
authors.forename: Xin-Shi
authors.initials: XS
authors.lastname: Zhang
authors.affiliation: Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China.|Capital Medical University School of Traditional Chinese Medicine, Beijing, 100069, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Ren
authors.affiliation: Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
authors.forename: Bao-Lin
authors.initials: BL
authors.lastname: Bian
authors.affiliation: Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
authors.forename: Hai-Yu
authors.initials: HY
authors.lastname: Zhao
authors.affiliation: Department of Pharmacy, Hebei North University, Zhangjiakou, 075000, China.
authors.forename: Shu
authors.initials: S
authors.lastname: Wang
mesh_terms: D000818:Animals; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D006801:Humans; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008862:Microsomes, Liver / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D012717:Sesquiterpenes / Q000032:analysis / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics
chemical_list: D011994:Recombinant Proteins; D012717:Sesquiterpenes; D003577:Cytochrome P-450 Enzyme System; C054337:tussilagone