Dimeric guaianolide sesquiterpenoids from the flowers of Chrysanthemum indicum ameliorate hepatic steatosis through mitigating SIRT1-mediated lipid accumulation and ferroptosis

The MS/MS-based molecular networking strategy led to the isolation of 27 new disesquiterpenoids from Chrysanthemum indicum . Chryindicolide O ( 15 ) ameliorated hepatic steatosis through mitigating SIRT1-mediated lipid accumulation and ferroptosis. • 27 new dimeric sesquiterpenoids were targeted isolated from the C. indicum flowers. • Chryindicolide O ameliorates hepatic steatosis in hepatocytes and zebrafish. • Chryindicolide O directly binds to and activates SIRT1. • Chryindicolide O reduces de novo lipogenesis and enhances fatty acid β-oxidation. • Chryindicolide O inhibits SIRT1-mediated ferroptosis. Non-alcoholic fatty liver disease (NAFLD) acts as the primary contributor to non-alcoholic steatohepatitis, fibrosis, cirrhosis, and potentially hepatocellular carcinoma. The flowers of Chrysanthemum indicum , a traditional edible medicinal herb, have been widely used in China for more than 2000 years. However, the function of C. indicum in managing NAFLD has seldom been investigated. To reveal the novel active components and underlying mechanisms of C. indicum in treating NAFLD. An MS/MS-based molecular networking-guided strategy was used for the chemical investigation. The structure identification of the new compounds involved high resolution electrospray ionization mass spectrometry (HRESIMS), 1D and 2D nuclear magnetic resonance (NMR) spectra, electronic circular dichroism (ECD), and X-ray crystallographic analysis. The biological evaluation was performed using Nile Red staining, flow cytometry, commercial kits, western blotting, co-immunoprecipitation, isothermal titration calorimetry, cellular thermal shift assay, drug affinity responsive target stability assay, molecular docking, and confocal immunofluorescence. A total of 27 new dimeric sesquiterpenoids, chryindicolides A-Z ( 1 - 26 ) and chrysanthemolide C ( 27 ), together with seven known compounds, were isolated from the flowers of C. indicum under the guide of MS/MS-based molecular networking. Among them, compounds 1 - 7 were rare chlorine-containing guaianolide dimers. Chryindicolide O (15) directly bound and activated the deacetylase Sirtuin 1 (SIRT1) to reduce de novo lipogenesis, enhance fatty acid β-oxidation, and inhibit ferroptosis in palmitic acid and oleic acid (P/O)-induced AML12 hepatocytes. In addition, chryindicolide O significantly ameliorated liver steatosis in high-fat diet-fed zebrafish. Novel guaianolide dimers from C. indicum alleviated hepatic steatosis through mitigating SIRT1-mediated lipid accumulation and ferroptosis, suggesting that they could be further developed as candidates against NAFLD.