Enzymatic Synthesis and Evaluation of Eight Methylated Quercetin Products: In Vitro Chemical Properties and Adipogenesis Regulation

The methoxylated modification of flavonoids has been reported to enhance stability and permeability; however, its effect on the improvement of activity is not clear. In this study, <i>Citrus depressa</i> flavonoid <i>O</i>-methyltransferase 5 and <i>Sorghum vulgare</i> 7-<i>O</i>-methyltransferase were recombinantly expressed and successfully converted quercetin (QUE) into eight methoxylated products, which were isolated and identified with a purity exceeding 95%. All products except rhamnetin (RHA) showed improved stability, while only 5,7,3',4'-EMQ, 7,3',4'-TMQ, and 3,7,3',4'-EMQ had higher uptake ratios. Compared to QUE, 5,7,3',4'-EMQ and RHA significantly reduced the intracellular triglyceride level, while 3,5,7,3',4'-PMQ, 3,3',4'-TMQ and 3,7,3',4'-EMQ increased it. 5,7,3',4'-EMQ and RHA also significantly downregulated both the mRNA and protein levels of peroxisome proliferator-activated receptor γ, while 3,5,7,3',4'-PMQ and 3,7,3',4'-EMQ upregulated PPARγ at the transcriptional level to about ten times higher than that of QUE. The structure-activity relationship analysis highlighted the importance of C3-OH retention and dual methoxylation of the A-ring. In summary, this study efficiently produced eight structurally well-defined QUE methoxylation products via biotransformation, established an in vitro initial structure-activity relationship for regulating adipogenesis, and provided a potential structure for PPARγ regulation, a central target of lipid metabolism.