Synthesis of Human Phase I and Phase II Metabolites of Hop (Humulus lupulus) Prenylated Flavonoids

Abstract

Hop prenylated flavonoids have been investigated for their in vivo activities due to their broad spectrum of positive health effects. Previous studies on the metabolism of xanthohumol using untargeted methods have found that it is first degraded into 8-prenylnaringenin and 6-prenylnaringenin, by spontaneous cyclisation into isoxanthohumol, and subsequently demethylated by gut bacteria. Further combinations of metabolism by hydroxylation, sulfation, and glucuronidation result in an unknown number of isomers. Most investigations involving the analysis of prenylated flavonoids used surrogate or untargeted approaches in metabolite identification, which is prone to errors in absolute identification. Here, we present a synthetic approach to obtaining reference standards for the identification of human xanthohumol metabolites. The synthesised metabolites were subsequently analysed by qTOF LC-MS/MS, and some were matched to a human blood sample obtained after the consumption of 43 mg of micellarised xanthohumol. Additionally, isomers of the reference standards were identified due to their having the same mass fragmentation pattern and different retention times. Overall, the methods unequivocally identified the metabolites of xanthohumol that are present in the blood circulatory system. Lastly, in vitro bioactive testing should be applied using metabolites and not original compounds, as free compounds are scarcely found in human blood.

The Pharmacokinetics of Individual Conjugated Xanthohumol Metabolites Show Efficient Glucuronidation and Higher Bioavailability of Micellar than Native Xanthohumol in a Randomized, Double‐Blind, Crossover Trial in Healthy Humans

“…Metabolites were found initially by using the correlation function in MS‐Dial when selecting reference compounds that were synthesized in previous work [ 15 ] by manually searching for glucuronic acid conjugates with an m/z of M+ GlcA (where M is the theoretical mass of XN/IXN or 8‐PN/6‐PN) and having the characteristic pharmacokinetic profile compared with XN‐7‐O‐ GlcA. Therefore, once all of the samples were uploaded, the MS1 spectra were compared for similarities in the abundance profile, but then, to confirm, the structures were tentatively annotated based on their MS2 spectra.…”

“…The analytes were separated using the LC method developed by Buckett et al (Supplementary information). [15] Each participant's samples were randomized and data were collected on a batch to batch within a time of 3 weeks.…”

“…[2] We recently published the synthesis of reference compounds for some XN metabolites, which now enables their identification and absolute quantification. [15] Therefore, the aim of the present study was to identify the main metabolites formed after oral consumption of native and micellar XN and investigate their pharmacokinetics individually.…”

“…[ 2 ] We recently published the synthesis of reference compounds for some XN metabolites, which now enables their identification and absolute quantification. [ 15 ]…”

An Updated Review of the Genus Humulus: A Valuable Source of Bioactive Compounds for Health and Disease Prevention

“…Pang et al (2007) reported that the poor oral bioavailability of this compound observed in vivo is mainly due to its specific binding to cytosolic proteins of intestinal epithelial cells and rapid metabolization by microorganisms in the colon [155,156]. Several studies have shown that, upon absorption, hop prenylflavonoids are rapidly metabolised into their corresponding glucuronide and sulphate derivatives, preventing the free forms from reaching the cells [156,157]. XN appears to show a biphasic absorption trend, with peak contraction observed between 1 and 7 h after ingestion, suggesting enterohepatic recirculation [156].…”