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.

Keywords: beer; blood analysis; hops; metabolites; prenylated flavonoids; synthesis; xanthohumol.

Figure 1

A generalised metabolic pathway of prenylated flavonoids from beer. Initial changes occur in the harsh environment of the gut, with additional metabolism by gut bacteria. Absorbed compounds that are changed in the gut (including xanthohumol) then make it to the liver, where active metabolism occurs.

Figure 2

The generalised reaction scheme (A) for the synthesis of prenylated flavonoid metabolites. Products 1–4 served as the scaffold to further react into certain metabolites in either chalcone or flavanone form [18,20]. (B) The structures of each prenylated flavonoid synthesised (see Supplementary Information for detailed structure elucidation data).

Figure 3

The DDA chromatogram (base shifted) of the analytical standards produced. Substances 1–17.

Figure 4

The identification of glucuronides after 60 min of XN consumption: co-chromatograms of the synthesised prenylated flavonoids (dashed blue, brown) and the compounds found in blood (green and gray).

Figure 5

Co-chromatogram of the synthesised XN/IXN sulfates (gray) and analysis of human plasma (orange); compounds 8 and 9 were tentatively assigned.

Figure 6

Co-chromatogram of the synthesised 8/6PN-sulfate, and then a human blood sample (green); compounds 12 and 15 were tentatively assigned.