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This study was aimed to examine the effect of apple phlorizin on lipid accumulation in HepG2 cell and hamsters (Thirty-six male Golden Syrian hamsters) fed with atherogenic diet. Dietary supplementation of 0.9% phlorizin significantly decreased the level of plasma total cholesterol (TC), triacylglycerols (TG), activity of cholesteryl ester transfer protein (CETP) and liver 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMG-CoA-R), alongside down-regulated the gene expressions of intestinal Niemann-Pick C1-like 1 (NPC1L1), HMG-CoA-R and up-regulated ATP-binding cassette transporters subfamily G members 5/8 (ABCG5/8) gene. Phlorizin increased the excretion of sterols, reduce absorption of dietary cholesterol and de novo synthesis of cholesterol. Furthermore, in silico studies revealed the binding profile of the phlorizin and HMG-CoA-R, hinted at the molecular interactions that are responsible for its activity. These results suggested that dietary supplementation of phlorizin could potentially possess cholesterol-lowering activity by increasing the excretion of sterols and mediating the endogenous cholesterol metabolic management.