Fatty liver disease is a condition in which abnormally large numbers of lipid droplets accumulate in liver cells. Fatty liver disease induces inflammation under conditions of oxidative stress and may result in cancer. To identify plants that protect against fatty liver disease, we examined the inhibitory effects of plant extracts on lipid droplet formation in mouse hepatoma cells. A screen of 98 water extracts of plants revealed 4 extracts with inhibitory effects. One of these extracts,
Fatty liver disease (FLD) is a disease in which abnormally large numbers of lipid droplets, mainly composed of triglyceride (TG), accumulate in liver cells. FLD is typically caused by excessive caloric consumption or alcohol intake. Nonalcoholic fatty liver disease (NFLD) is FLD not caused by alcohol consumption and its prevalence is estimated to be 20–30% of the population in Western countries [
Lipid droplets in liver cells are intracellular storage sites for neutral lipids mainly composed of TG [
In the present study we report the identification of plant extracts that inhibit lipid droplet formation in liver cells
Gallic acid monohydrate, ellagic acid dihydrate, rubusoside, olive oil (suitable for lipase assay), and the TG E-Test Wako kit were purchased from Wako (Tokyo, Japan). Most of dried plants including dried
The mouse hepatoma cell line Hepa 1–6 was obtained from RIKEN Cell Bank. The cells were grown in humidified 5% CO2 at 37°C in DMEM containing 10% fetal bovine serum.
Dried plants were ground with a Labo Milser ML-2 (Iwatani Corporation, Osaka, Japan) and 0.05 g of the resulting powder was extracted with 500
Lipid droplets were formed in hepatoma cells using the methods of Fujimoto et al. [
Hepa 1–6 cells (
Cell growth assay was performed according to Kueng et al. [
Total protein was assayed using the BCA protein assay reagent kit according to the manufacturer’s instructions (Pierce, IL, USA).
Animal experiments were performed according to international ethics standards and animal protocols were approved by the Niigata University of Pharmacy and Applied Life Sciences (Niigata Japan). The use of animals complied with the guidelines established by the Animal Care Committee of Niigata University of Pharmacy and Applied Life Sciences. A normal diet (CLEA Rodent Diet CE-2) and a high-fat diet containing 32% fat by weight (HFD 32) were purchased from CLEA Japan, Inc. (Tokyo, Japan). RSE used in animal experiments was prepared as follows. Dried
Liver tissues were excised, rinsed with cold PBS, and homogenized in 3 volumes of water using a BioMasher II (Nippi, Tokyo, Japan). Total lipids were extracted from 280
Mouse liver samples were collected, fixed in 10% formalin buffered solution, and then embedded in OCT compound, frozen in liquid nitrogen, and sectioned (5
To screen plants for the ability to prevent NAFLD, we assessed the inhibitory activity of the extracts on lipid droplet formation induced by oleic acid in the mouse hepatoma cell line Hepa 1–6. We chose Hepa 1–6 cells because these cells contain a small number of lipid droplets when cultured in standard medium such as DMEM supplemented with 10% FCS (Figure
Inhibitory effects of plant extracts (5%) on lipid droplet formation in Hepa 1–6 cells treated with oleic acid. Dried plants were ground with a mill and 0.05 g of the resulting powder was extracted with 500
Lipid droplet formation in hepatoma cells treated with oleic acid and inhibition of lipid droplet formation by RSE. The mouse hepatoma cell line Hepa 1–6 was used for the experiments. Dried
Among these, we focused on
Effect of plant extracts on cell growth. Hepa 1–6 cells (
We next analyzed TG content in oleic acid- and RSE-treated Hepa 1–6 cells. Cellular lipids were extracted, separated by silica-gel chromatography (TLC), and visualized with cupric acetate. Analysis of intracellular lipids revealed a dramatic increase in TG content in oleic acid-treated Hepa 1–6 cells, whereas no oleic acid was detected in the cells (Figure
Effect of RSE on intracellular TG content in Hepa 1–6 cells. (a) TG content in RSE-treated Hepa 1–6 cells. Dried
Effect of gallic acid on lipid droplet formation in Hepa 1–6 cells. The cells (20,000 cells/well) were seeded on a 96-well microtiter plate and incubated overnight. The next day, 0.6 mM oleic acid conjugated to BSA and gallic acid (250
To determine if RSE is also effective
Body weight gain of mice fed HFD and treated with RSE. Body weight was measured weekly. Body weight gain was greater in HFD-fed mice than in those fed a normal diet. RSE extract had no significant effect on body weight gain in HFD-fed mice (
The livers of the normal diet-fed mice were a healthy red color, whereas those of HFD-fed mice had a white color due to TG accumulation. The livers of HFD-fed and RSE-treated mice (HFD + RSE) partially retained a red color (Figure
Effect of RSE on mouse fatty liver. (a) Mouse liver. As shown in the figure, the livers of normal diet-fed mice had a healthy red color, whereas those of HFD-fed mice had a white color due to TG accumulation. The livers of HFD-fed RSE-treated mice (HFD + RSE) partially retained their red color. (b) TG content in mouse liver. TG content in mouse liver was measured using the TG E-Test Wako kit. Data are the means of six experiments (
Histological detection of mouse NAFLD. Frozen sections of liver were prepared and after fixation, intracellular lipid droplets were stained red with Oil Red O. Nuclei were stained blue with hematoxylin staining to identify individual cells. Livers from a mouse fed a normal diet and mice fed a high-fat diet with (HFD + RSE) or without RSE (HFD) are shown. In the liver sections of HFD-fed mice, many red-stained lipid droplets were observed, whereas only a small number of lipid droplets were seen in the livers of mice fed a normal diet. The number and size of lipid droplets were much lower in the livers of RSE-treated mice (HFD + RSE).
A screen of 98 plant extracts identified 4 extracts with inhibitory effects on lipid droplet formation in a cell culture assay. Those were
Gallic acid is a strong candidate for the effects of RSE on TG since its antisteatotic effect in rats and mice was reported previously [
In the present study we successfully identified a plant extract, RSE, which prevented NAFLD in cell culture and animal models. Our studies suggest that RSE, also known as Tien-cha and widely consumed in China and Japan as a beverage, shows promise for the prevention or treatment of NAFLD.
Triglyceride
Thin-layer chromatography
Fatty liver disease
Nonalcoholic fatty liver disease.
The authors declare that there is no conflict of interests regarding publication on this paper.
The authors thank Masato Abe, Tasuku Nakabayashi, Yu Sakurai, Masashi Watanabe, and Sho Motomiya for technical assistance. This study was supported in part by a Strategic Research Foundation Grant-Aided Project for Private Universities grant from the Ministry of Education, Culture, Sport, Science, and Technology, Japan (MEXT), 2010–2014 (S1001030).