3,5,6,7,8,3′,4′-Heptamethoxyflavone (HMF), one of the highest O-methyl numbers in the polymethoxyflavones, possesses various significant health benefits. However, the in vivo metabolic profile of HMF remains largely unexplored. In this study, a systematic identification and relative quantitation of HMF and its metabolites in rats were performed using UHPLC-LTQ-Orbitrap mass spectrometry combined with enzymatic hydrolysis. A total of 56 metabolites were tentatively identified, including 37 demethylated metabolites and 19 glucuronide conjugates of these demethylated products. Notably, all seven mono-demethylated metabolites of HMF were simultaneously detected and structurally discriminated for the first time in rats, along with other di-, tri-, tetra-, and penta-demethylated metabolites. Semi-quantitative analysis revealed that HMF underwent predominant biotransformation into mono-demethylated metabolites in rats, followed by progressive sequential demethylation of these metabolites to generate more di-, tri-, tetra-, and penta-demethylated metabolites. Demethylation and glucuronidation are the primary metabolic pathways of HMF in vivo. This study presents the first comprehensive elucidation of the in vivo metabolic profile of HMF, which is helpful for further understanding of its in vivo potential effective components and pharmacological mechanism.

Dietary components play a critical role in modulating gut microbiota composition. Pectin has a positive regulatory effect on the disorder of gut microbiota-metabolites caused by a high-fat diet. A cyclic low-fat/high-fat diet (CLHFD), the alternating cycle between short-term low-fat and high-fat diets (HFD), capitalizes on the rapid adaptability of gut microbiota to short-term HFD interventions. Building on this plasticity, the effect of CLHFD on the composition of the gut microbiota and bile acids (BAs) with and without apple pectin (AP) treatment have attracted our attention. The experimental results showed that CLHFD has the higher Acetatifactor abundance than low-fat diet LFD, while AP selectively enriched Duncaniella and Bacteroides under CLHFD. Notably, AP upregulated hepatic CYP7A1 expression and mitigated CLHFD-induced BA metabolic abnormalities. Our results position AP as a microbiota-modulating supplement to buffer the metabolic stress of dietary shifts, with potential applications in personalized nutrition programs.