Optimized extraction of polymethoxyflavonoids from Citrus reticulate ‘Chachi’ peel and their anti-atherosclerotic potential

Polymethoxyflavonoids (PMFs), a featured phytochemical group derived from Citrus reticulata ‘Chachi’ (GCP), have demonstrated potential therapeutic benefits for cardiovascular disease. In the present study, we developed an efficient method for extracting PMFs from GCP using HP20 macroporous resin. The extraction protocol was optimized using response surface methodology and systematically evaluated by liquid chromatography-high resolution mass spectrometry (LC-HRMS). Scale-up experiments performed in triplicate confirmed the robustness of the method, achieving PMF yields of 94 % with a purity of 72 %. The anti-atherosclerotic effects of PMFs were evaluated using a hyperlipid feed (HLF)-induced ApoE −/− mouse model. PMFs treatment significantly reduced lipid plaques in the aorta, decreased hepatic lipid accumulation, and improved serum lipid profiles. PMFs enhanced the antioxidant defense system by reducing malondialdehyde (MDA) levels while upregulating superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities. Lipidomics analysis demonstrated that PMFs ameliorated hepatic lipid disorders and reduce the generation of oxidized phospholipids. Further investigations revealed that PMFs suppressed reactive oxygen species (ROS) production and CD36 expression in the aortic root, downregulated hepatic CD36 expression and upregulate SR-B1, ABCG5 and ABCG1 expression to maintain cholesterol homeostasis. In conclusion, this study provides the first direct evidence that PMFs inhibit the early arterial plaque formation in vivo. Additionally, our findings suggested that the anti-atherosclerotic effect of PMFs may involve ROS/lipid peroxidation/ CD36 mechanism. These results highlight PMFs as promising candidates for atherosclerosis prevention and treatment, emphasizing their multi-target pharmacological actions. • An effective method was developed to extract Polymethoxyflavonoids from C. reticulate . • The high efficiency of the method was evaluated by LC-MS. • PMFs reduce aroma plaques, alleviate oxidative stress and inflammation levels. • PMFs exert the Atherosclerosis via ROS/lipid peroxidation/CD36 axis.