Anti-Adipogenic Effects of Ethanol Extracts Prepared from Selected Medicinal Herbs in 3T3-L1 Cells

Abstract

Obesity is a major risk factor for various metabolic diseases such as cardiovascular disease, hypertension, and type 2 diabetes mellitus. In this study, we prepared ethanol extracts from Agastache rugosa (ARE), Chrysanthemum zawadskii (CZE), Mentha arvensis (MAE), Perilla frutescens (PFE), Leonurus sibiricus (LSE), Gardenia jasminoides (GJE), and Lycopus coreanus (LCE). The anti-oxidant and anti-adipogenic effects were evaluated. The IC₅₀ values for ascorbic acid and LCE against 2,2-diphenyl-1-picrylhydrazyl radicals were 246.2 μg/mL and 166.2 μg/mL, respectively, followed by ARE (186.6 μg/mL), CZE (198.6 μg/mL), MAE (337.1 μg/mL), PFE (415.3 μg/mL), LSE (548.2 μg/mL), and GJE (626.3 μg/mL). In non-toxic concentration ranges, CZE had a strong inhibitory effect against 3T3-L1 adipogenes (84.5%) than those of the other extracts. Furthermore, the anti-adipogenic effect of CZE is largely limited in the early stage of adipogenesis, and we revealed that the inhibitory role of CZE in adipogenesis is required for the activation of Wnt signaling. Our results provide scientific evidence that the anti-adipogenic effect of CZE can be applied as an ingredient for the development of functional foods and nutri-cosmetics for obesity prevention.

Keywords: Chrysanthemum zawadskii; Wnt signaling; adipognesis; medicinal herb; obesity.

Fig. 1

Scheme of 3T3-L1 differentiation and the treatment of extracts from medicinal herbs. The 3T3-L1 cells were treated with ethanol extracts of medicinal herbs during day −2 to day 6. DM, differentiation medium consist of fetal bovine serum (FBS)-Dulbecco’s modified Eagle’s medium (DMEM) including 3-isobutyl-1-methylxanthine (500 μM), dexamethasone (5.2 μM), and insulin (167 nM); Post-DM, post-differentiation medium consist of FBS-DMEM including insulin (167 nM).

Fig. 2

Effects of ethanol extracts from medicinal herbs treatment on the adipogenesis of 3T3-L1 cells. The 3T3-L1 cells were treated with 10 and 50 μg/mL concentration in the presence or absence of differentiation media during day −2 to day 6. The size and number of lipid droplets were observed (A). The levels of lipid accumulation were quantified by Oil red O staining using microplate reader at 510 nm (B). Significant differences by one-way ANOVA followed by Student’s t-tests (*P<0.05, **P<0.01, and ***P<0.001 versus Adi). Pre, 3T3-L1 preadipocytes; Adi, mature 3T3-L1 adipocytes, ARE, Agastache rugose ethanol extract; CZE, Chrysanthemum zawadskii ethanol extract; MAE, Mentha arvensis ethanol extract; PFE, Perilla frutescens ethanol extract; LSE, Leonurus sibiricus ethanol extract; GJE, Gardenia jasminoides ethanol extract; LCE, Lycopus coreanus ethanol extract.

Fig. 3

Effects of Chrysanthemum zawadskii ethanol extract (CZE) treatment of various stages on the lipid accumulation of 3T3-L1 cells. The CZE treatment were divided into four stages, pre-adipogenic stage (a: day −2~0), early stage (b: day 0~2), intermediate stage (c: day 2~4), and terminal stage (d: day 4~6). (A) Levels of lipid accumulation were evaluated by ORO staining and quantified using microplate reader at 510 nm. (B) The number and size of lipid droplets were observed using inverted microscope. Significant differences by one-way ANOVA followed by Student’s t-tests (***P<0.001 versus Adi). Pre, 3T3-L1 preadipocytes; Adi, mature 3T3-L1 adipocytes.

Fig. 4

The Chrysanthemum zawadskii ethanol extract (CZE) treatment downregulate mRNA levels of adipogenic transcriptional factors in 3T3-L1 cells. The mRNA expression levels of adipogenic transcriptional factors such as CCAAT/enhancer-binding protein (C/EBP)β (A), C/EBPα (B), peroxisome proliferator-activated receptor (PPAR) γ (C), and fatty acid synthase (FAS) (D) were estimated by RT-PCR analysis. β-Actin was used as a control. Pre, 3T3-L1 preadipocytes; Adi, mature 3T3-L1 adipocytes.

Fig. 5

The anti-adipogenic effect of Chrysanthemum zawadskii ethanol extract (CZE) is required the Wnt signaling. 3T3-L1 cells were exposed to 50 μg/mL of CZE in the presence or absence of 10 μM 4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-benzamide (IWR-1) from day −2 to day 2. Intracellular lipid accumulation was visualized using Oil red O staining. Pre, 3T3-L1 preadipocytes; Adi, mature 3T3-L1 adipocytes.