Z. schinifolium was obtained from Dongeui Oriental Hospital, Dongeui University College of Korean Medicine (Busan, Republic of Korea). Dried leaves from Z. schinifolium (40 g) were cut into small pieces, ground into a fine powder and then soaked with 500 ml 100% ethanol (Sigma-Aldrich, St. Louis, MO, USA) for 2 days. The extracted liquid was filtered through Whatman No. 3 filter paper (Sigma-Aldrich) twice to remove any insoluble materials and was then concentrated using a rotary evaporator (EYELAN-1000; Rikakikai Co., Ltd., Tokyo, Japan). The extracts were redissolved in dimethylsulfoxide (DMSO; Sigma-Aldrich) to a final concentration of 200 mg/ml (extract stock solution) and were subsequently diluted with Dulbecco’s modified Eagle’s medium (DMEM; WelGENE Daegu, Korea), to the desired concentration prior to use.

The 3T3-L1 cell line was obtained from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in DMEM, supplemented with 10% fetal bovine serum (FBS; WelGENE) and 1% penicillin-streptomycin (Sigma-Aldrich) at 37°C in a humidified atmosphere of 5% CO₂. To initiate differentiation of the 3T3-L1 pre-adipocytes into adipocytes, the cells were stimulated with differentiation inducers, including 0.5 mM isobuthylmethylxanthine (Sigma-Aldrich), 10 μg/ml insulin (MDI; Sigma-Aldrich) and 1 μM dexamethasone, which were added to the DMEM, containing 10% FBS, for 48 h. Following differentiation, the adipocytes were incubated with post-differentiation medium, which consisted of DMEM, containing 10% FBS and 10 μg/ml insulin. The medium was replaced every other day for up to 8 days (29).

To assess the cell viability in the 3T3-L2 adipocytes, the cells were plated into 24-well plates (1×10⁵ cells/ml) and incubated for 24 h at 37°C until confluent. The cells were subsequently treated with the indicated concentrations (100–600 μg/ml) of EEZS for 72 h at 37°C. The control cells were supplemented with complete medium, containing 0.5% DMSO, as a vehicle control. Subsequent to incubation for 72 h at 37°C, the medium was removed and the cells were incubated with 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) solution for 3 h at room temperature. The supernatant was subsequently discarded and the formazan blue, which had formed in the cells, was then dissolved in 100% DMSO. The DMSO concentration did not exceed 0.05%. The optical density was measured at 540 nm using a microplate reader (Dynatech Laboratories, Chantilly VA, USA) and the effects of EEZS on the inhibition of cell growth were assessed as the percentage of cell viability, in which the vehicle-treated cells were considered to be 100% viable.

The intercellular lipid accumulation within adipocytes was assessed using Oil Red-O solution. Following the induction of adipocyte differentiation, the cells were washed twice with phosphate-buffered saline (PBS; WelGENE) and fixed at room temperature with 10% formalin (Junsei Chemical Co., Ltd., Tokyo, Japan) for 1 h. The cells were dried and stained with Oil Red-O (0.35% Oil Red-O in 100% aqueous 2-isopropanol; Sigma-Aldrich) for 20 min at room temperature. Following staining of the lipid droplets, the Oil Red-O staining solution was removed, the plates were rinsed twice with PBS and 60% isopropanol and were then dried. Images of the stained oil droplets in the 3T3-L1 cells were captured under light microscopy. To quantify the intracellular lipids, the stained lipid droplets were dissolved in isopropanol, containing 4% Nonidet P-40 (NP-40; Sigma-Aldrich). The extracted dye was transferred into a 96-well plate, and the absorbance was measured using a microplate reader (MR5000; Dynatech Laboratories, Chantilly, VA, USA) at 500 nm. The difference in absorbance between the samples with and without dye solution was calculated (30).

The cells were harvested and washed once with ice-cold PBS, and were gently lysed for 20 min in ice-cold lysis buffer containing 40 mM Tris (pH 8.0), 120 mM NaCl, 0.5% NP-40, 0.1 mM sodium orthovanadate, 2 μg/ml leupeptin and 100 μg/ml phenylmethylsulfonyl fluoride (all Sigma-Aldrich). The supernatants were collected and the protein concentrations were determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Equal quantities of the protein extracts were denatured by boiling at 95°C for 5 min in sample buffer (Bio-Rad Laboratories, Inc.), containing 0.5 M Tris-HCl (pH 6.8), 4% sodium dodecyl sulphate (SDS), 20% glycerol, 0.1% bromophenol blue and 10% β-mercaptoethanol, at a ratio of 1:1. The samples were either stored at −80°C or were used immediately for immunoblotting. Aliquots, containing 30 μg total protein, were separated on 10% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (Amersham Life Sciences, Arlington Heights, IL, USA). The membranes were subsequently blocked with 5% non-fat milk and incubated overnight at 4°C with primary antibodies, probed with enzyme-linked secondary antibodies at room temperature for a further 1 h and then detected using an enhanced chemiluminescence detection system (Amersham Life Sciences), according to the manufacturer’s instructions. The primary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) and Cell Signaling Technology, Inc. (Danvers, MA, USA) (Table I). The horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG, sc-2004; 1:1,000), anti-mouse IgG (sc-2005; 1:1,500) and anti-goat IgG (sc-2350; 1:1,500) were purchased from Santa Cruz Biotechnology, Inc. β-actin was used as an internal control.

The data are expressed as the mean ± standard deviation. Comparisons between the groups were made using analysis of variance and the significance between differences were analyzed using Duncan’s multiple range test. Statistical analyses were performed using SPSS version 18.0 (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistical significant difference.

To determine the cytotoxicity of EEZS, the 3T3-L1 cells were treated with various concentrations of EEZS and the cell viability was assessed using an MTT assay. The data demonstrated that EEZS exhibited no cytotoxic effects at concentrations ≤200 μg/ml (Fig. 1). Therefore, concentrations of 50, 100, 150 and 200 μg/ml were selected for use in the subsequent experiments.

As increased lipid accumulation during the differentiation of pre-adipocytes into adipocytes is a typical phenomenon, which occurs in 3T3-L1 cells and is used as a marker of differentiation, the 3T3-L1 pre-adipocytes were treated with various concentrations of EEZS in the presence of MDI or with MDI alone for 8 days. Lipid accumulation was subsequently measured in the cells using Oil Red-O staining. As shown in Fig. 2, oil droplets were not visible in the undifferentiated 3T3-L1 cells, however, several lipid droplets were visible in the fully differentiated cells treated with MDI. Microscopic observations of the Oil Red-O staining revealed a reduction in the number of lipid droplets as the concentrations of EEZS increased. The inhibitory effects of EEZS on the triglyceride contents of the differentiated adipocytes were measured, which revealed that the triglyceride contents of the adipocytes increased markedly during 8 days incubation with MDI (Fig. 3). However, treatment with EEZS decreased the triglyceride levels significantly, in a concentration-dependent manner. These results demonstrated that EEZS inhibited adipocyte differentiation in the 3T3-L1 cells at non-cytotoxic concentrations.

Adipogenesis is accompanied by the increased expression of adipogenic transcription factors and adipocyte-specific genes. To investigate the anti-adipogenic mechanism underlying EEZS, the fully differentiated adipocytes were treated with different concentrations of EEZS, and the protein expression levels of PPARγ, C/EBPα and C/EBPβ were determined by western blot analysis. As expected, the levels of these three proteins were significantly upregulated during the process of differentiation (Fig. 4). However, treatment with EEZS suppressed the expression levels of PPARγ, C/EBPα and C/EBPβ compared with the fully differentiated control adipocytes, and this occurred in a concentration-dependent manner. This result suggested that EEZS inhibited adipogenesis by reducing the expression of C/EBPβ, which lead to a downregulation in the expression levels of C/EBPα and PPARγ.

The control of adipogenesis requires two well-established signaling mechanisms, the ERK and PI3K/Akt pathways, which are important upstream of adipocyte differentiation (17). The 3T3-L1 cells were pretreated with either ERK- or PI3K-specific inhibitors for 1 h prior to incubation with MDI in the presence or absence of 200 μg/ml EEZS, and the levels of phosphorylated-ERK, PI3K and Akt were determined at various time-points by western blotting. Consistent with previous data (16,18), the expression of phosphorylated-ERK was significantly increased during the early stages of adipogenesis, and the activation continued for 3 h following the induction of adipocyte differentiation by DMI (Fig. 5A). However, treatment with EEZS effectively suppressed the MDI-induced phosphorylation of ERK. The specific ERK inhibitor, PD98059, induced significant inhibition of the phosphorylation of ERK and used as a positive control for treatment with EEZS (Fig. 5B). As shown in Fig. 6, treatment with DMI rapidly induced the phosphorylation of PI3K and Akt, and this continued for 6 h subsequent to treatment. However, the levels of phosphorylated-PI3K and Akt were markedly attenuated following treatment with EEZS, similar to the results, which were obtained using LY294002, a PI3K-specific inhibitor. These results suggested that the inhibition of adipocyte differentiation by EEZS was associated with inactivation of the ERK and PI3K/Akt signaling pathways.