Molecular Mechanism of Mulberrin Regulating Lipid Metabolism Homeostasis Based on the Gut–Liver Axis Fiaf/AMPK Signaling Pathway

ABSTRACT Lipid metabolism disorders and the induced metabolic diseases have risen to become one of the primary public health issues. Our preliminary experimental results confirmed that mulberry leaf flavonoid extract exhibited efficacy in lipid metabolism regulation; the present research further explored its material basis and molecular mechanism. First, ELISA methylglutaryl‐CoA reductase (HMGCR) activity was employed as an in vitro evaluation index. The result indicated that the main flavonoids in mulberry leaves, including mulberrin (Mul), cyclomulberrin, morusin, and morin, exhibited strong activity, while Mul, obtaining two isopentene groups in C‐3 and C‐8 positions, exhibited the best activity with an IC 50 of 2.21 µg/mL. Molecular docking indicated that the HMGCR_Mulberrin complex had the lowest binding energy and formed the most hydrogen bonds. Animal experiments further confirmed the positive regulatory effect of Mul on lipid metabolism, reducing body weight and TC and TG levels in hyperlipidemic C57BL/6 mice. Besides, the obesity related Firmicutes / Bacteroidetes value in gut microbiota was significantly inhibited under the Mul treatment, among these, the relative abundance of Lachnospiraceae decreased, while Lactobacillus increased significantly. Differential metabolite analysis indicated the production of quercetin 3'‐sulfate, quercetin 3‐sulfate, morin, Swainsonine, and isoferulic acid in urine or feces with bioactivity. Combined with the KEGG pathway analysis, as well as validation via RT‐qPCR and western blotting, our findings demonstrated that Mul activated the fasting‐induced adipocyte factor (Fiaf)/ adenosine monophosphate‐activated protein kinase (AMPK) signaling pathway through the gut–liver axis, thereby exerting a regulatory role in lipid metabolism. These results provide a theoretical basis and novel approach for developing new hypolipidemic functional foods.