Anti-obesity activity, acute toxicity, and chemical constituents of aqueous and ethanol Viola mandshurica extracts

Abstract

Background: Viola mandshurica has traditionally been used as an expectorant, diuretic, and anti-inflammatory drug. The present study was designed to test the hypothesis that low doses of two different V. mandshurica extracts have anti-obesity effects.

Methods: We evaluated the effects of ethanol extract (VME) and aqueous extract (VMA) from V. mandshurica on high-fat diet (HFD)-induced obese mice as well as the acute oral toxicities and chemical compositions of both extracts.

Results: Oral administration of VME or VMA (50, 100, or 200 mg/kg) decreased body weight gain, liver and adipose tissue mass, adipocyte size, and serum lipid levels. Both extracts increased adiponectin serum concentrations and mRNA expression in epididymal adipose tissue. VME and VMA also reversed the HFD-induced mRNA expression of lipogenic genes such as CCAAT/enhancer binding protein (C/EBP)α, C/EBPβ, sterol regulatory element-binding protein 1c, and leptin in adipose tissue, whereas they increased mRNA expression of uncoupling protein 2 and adenosine monophosphate-activated protein kinase (AMPK). VME and VMA increased the phosphorylation of AMPK and acetyl-coA carboxylase with a concomitant decrease in fat accumulation in the liver. High performance liquid chromatography analysis revealed that both VME and VMA contained esculetin (0.566% for VME, 0.231% for VMA) and schaftoside (0.147% for VME, 0.126% for VMA). In a 2-week acute toxicity study, administration of a single oral dose of VME or VMA (5000 mg/kg) caused no signs of toxicity or mortality.

Conclusions: These results suggest that both VM extracts exert anti-obesity effects in HFD-induced obese mice by suppressing lipogenesis and activating AMPK in the liver and adipose tissue. Our findings suggest that VM extracts could be a safe and effective treatment for obesity.

Keywords: AMP-activated protein kinase; Adiponectin; Adipose tissue; Liver; Toxicity.

Fig. 1

Effects of VME and VMA on HFD-induced obese mice. a-b Body weight, c FER, d fat weight, and e liver, kidney, and spleen tissue weights. Data are presented as mean ± SEM (n = 12); #p < 0.05, ##p < 0.01, ###p < 0.001 vs. ND-normal; *p < 0.05, **p < 0.01, ***p < 0.001 vs. HFD-control

Fig. 2

Effects of VME and VMA on serum lipid levels in HFD-induced obese mice. a Triglyceride, b free fatty acid, c glucose, d total cholesterol, e LDL-cholesterol, and f HDL-cholesterol levels. Data are presented as mean ± SEM (n = 12); #p < 0.05, ##p < 0.01, ###p < 0.001 vs. ND-normal; *p < 0.05, **p < 0.01, ***p < 0.001 vs. HFD-control

Fig. 3

Effects of VME and VMA on serum biochemical parameters in HFD-induced obese mice. a Creatinine, b AST and ALT, c leptin, and d adiponectin levels. Data are presented as mean ± SEM (n = 6–12); #p < 0.05, ##p < 0.01, ###p < 0.001 vs. ND-normal group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. HFD-control

Fig. 4

Effects of VME and VMA on histology of adipose tissue in HFD-induced obese mice. a Epididymal adipose tissue morphology and b adipocyte area. Representative images of H&E-stained sections. Data are presented as mean ± SEM (n = 4); #p < 0.05, ##p < 0.01, ###p < 0.001 vs. ND-normal; *p < 0.05, **p < 0.01, ***p < 0.001 vs. HFD-control

Fig. 5

Effects of VME and VMA on mRNA levels in epididymal adipose tissue. a C/EBPα, b C/EBPβ, c SREBP1c, d UCP2, e AMPKα1, f AMPKα2, g leptin, and h adiponectin. Data are presented as mean ± SEM (n = 4); #p < 0.05, ##p < 0.01, ###p < 0.001 vs. ND-normal; *p < 0.05, **p < 0.01, ***p < 0.001 vs. HFD-control

Fig. 6

Effects of VME and VMA on liver morphology in HFD-induced obese mice. Tissue sections stained with a H&E and b Oil Red O

Fig. 7

Effects of VME and VMA on hepatic AMPK expression and HPLC chromatogram. a AMPKα1 and AMPKα2 mRNA expression and b AMPK and ACC phosphorylation. c HPLC-PDA chromatograms of two standards mixture, VME, and VMA at 330 nm. Esculetin and schaftoside appeared at retention times of approximately 12.3 and 21.7 min, respectively. Data are presented as mean ± SEM (n = 4); #p < 0.05, ##p < 0.01, ###p < 0.001 vs. ND-normal; *p < 0.05, **p < 0.01, ***p < 0.001 vs. HFD-control