Identification of Plants That Inhibit Lipid Droplet Formation in Liver Cells: Rubus suavissimus Leaf Extract Protects Mice from High-Fat Diet-Induced Fatty Liver by Directly Affecting Liver Cells

Figure 3

Effect of plant extracts on cell growth. Hepa 1–6 cells () were suspended in 100 μL DMEM supplemented with 10% FCS and seeded in each well of a 96-well plate and incubated for 24 h. After incubation, the medium was replaced with 100 μL DMEM containing 10% FCS, 0.6 mM oleic acid conjugated with 0.12 mM defatted bovine serum albumin (BSA) and plant extract and further incubated for 12 or 24 h. After incubation, the cells were fixed with 0.25% glutaraldehyde for 5 min. The cells were then washed with phosphate-buffered saline (PBS) and stained with 0.1% crystal violet for 15 min. After the staining, 0.1% crystal violet was removed and the cells were washed with tap water. After the washing, 100 μL of 10% acetic acid was inoculated in each well and mixed. A portion of the resulting solution (33 μL) was transferred to each well of a new 96-well plate. To this, 64 μL of 10% acetic acid was added and mixed. Absorbance (570 nm) of each sample was measured by Microplate Reader Model 550 (Bio-Rad, Tokyo, Japan). NJE: Nuphar japonicum rhizome extract. Data are means of five experiments presented with SD bars.