Identification of Polymethoxyflavones (PMFs) from Orange Peel and Their Inhibitory Effects on the Formation of Trimethylamine (TMA) and Trimethylamine-N-oxide (TMAO) Using cntA/B and cutC/D Enzymes and Molecular Docking

Abstract

This study investigates the inhibitory effects of polymethoxyflavones (PMFs) on enzymes involved in the production of trimethylamine (TMA) and trimethylamine-N-oxide (TMAO). PMFs were isolated from Valencia orange peel and identified using column separation and NMR techniques. The findings reveal that nobiletin and 3,6,7,8,2′,5′-hexamethoxyflavone significantly suppress cntA/B and cutC/D, respectively. Furthermore, 3,6,7,8,2′,5′-hexamethoxyflavone decreases the level of TMAO formation by suppressing the FMO3 mRNA level. This study elucidates that specific structural features of PMFs can contribute to their interactions with enzymes. Our study represents the first demonstration of the ability of PMFs to mitigate the risk of cardiovascular disease (CVD) by inhibiting enzymes responsible for TMA production, which are generated by gut microbiomes. Furthermore, we introduce a novel model system utilizing TMA-induced HepG2 cells to assess and compare the inhibitory effects of PMFs on TMAO production. These findings could pave the way for the development of novel therapeutic approaches to manage CVD.

Discovery of a Novel Bioactive Compound in Orange Peel Polar Fraction on the Inhibition of Trimethylamine and Trimethylamine N-Oxide through Metabolomics Approaches and In Vitro and In Vivo Assays: Feruloylputrescine Inhibits Trimethylamine via Suppressing cntA/B Enzyme

“…23 Additionally, 3,6,7,8,2′,5′-hexamethoxyflavone effectively suppressed FMO3 mRNA expression and TMAO formation. 23 Polymethoxyflavones-rich fraction markedly enhanced Bif idobacterium and Lactobacillus, indicating a health-promoting activity by modulating the gut microbiota. 24 Citrus peel decreased the TMAO production through altering gut microbiota composition and reducing FMO3 expression level.…”

“…The condition of Ultimate 3000 LC system coupled to a TSQ Quantiva triple quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose, CA) was shown in our previous study. 23 The limits of detection minimum concentration on the basis of the signal-to-noise (S/N) ratio of 3 and 10, respectively (Table S1).…”

“…Moreover, the inhibitory effect of OPNP on cntA/B enzyme and TMA production is attributed to the presence of polymethoxyflavones. 23 These findings represent a significant shift in our understanding of the beneficial compounds present in orange peel. While previous research had focused on polymethoxyflavones, such as nobiletin, as the primary bioactive compounds, this study has unveiled a new perspective.…”

“…Our recent study reported that the treatment of nobiletin, 5-hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone, and sinesetin was effective in the inhibition of cntA/B enzyme activity and TMA formation. 23 Polymethoxy- flavones were more abundant in OPNP, compared to that in OPP, due to the hydrophobic properties of compounds. The concentrations of nobiletin, 5-hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone, and sinesetin in OPNP were 3149.4 ± 542.2 μg/g of extract, 167.5 ± 47.6 μg/g of extract, and 2090.7 ± 188.0 μg/g of extract, respectively.…”

“… ,, Additionally, phenolamides like feruloylputrescine (FP) are found in citrus plants and formed through the decarboxylation of l -arginine, and they can help reduce the risk of various CVDs . Our previous study has shown that nobiletin isolated from orange peel was the most effective in reducing TMA levels by inhibiting cntA/B enzyme . Additionally, 3,6,7,8,2′,5′-hexamethoxyflavone effectively suppressed FMO3 mRNA expression and TMAO formation .…”