Cell culture reagents were obtained from Life Technologies (Grand Island, NY, USA). Anti-C/EBPα and anti-C/EBPβ antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-PPARγ antibody and secondary antibody were purchased from Cell Signaling (Beverly, MA, USA). Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

All animals were obtained from the Korea Research Institute of Bioscience and Biotechnology, Daejoen, Korea. Seven-week-old C57BL/6 male mice were subdivided, acclimatized for 1 week, and divided randomly into five groups: (1) sedentary, normal diet, and vehicle treatment (ND-Veh), (2) sedentary, high-fat diet, and vehicle treatment (HFD-Veh), (3) sedentary, high-fat diet, and THI treatment (HFD-THI), (4) exercise, high-fat diet, and vehicle treatment (E/HFD-Veh), and (5) exercise, high-fat diet, and THI treatment (E/HFD-THI). Bedding was changed once a week, and the temperature and humidity were controlled. Mice were housed under 12 hr light/12 hr dark conditions. The plans and protocols for animal experiments were approved by the Institutional Animal Care and Use Committee of Sookmyung Women's University, Seoul, Korea.

The roots of Platycodon grandiflorum and Scutellariae baicalensis were obtained from Namil Farm & Ginseng Co. (Geumsam, Korea); voucher specimens were preserved at Chug-Ang University College of Medicine, Seoul, Korea (No. 2009.11-12). The two herbs were mixed in a 1:1 ratio and extracted with 70% ethanol. The extract was concentrated by evaporating solvent under low pressure conditions. The final yield of extract compared with raw herbal material was 25% (w/w).

Purina Diet (Koatech, Seoul, Korea) was provided to mice in the normal diet group, whereas a pellet rodent diet with 60% K cal fat (Central Lab. Animal Inc., Seoul, Korea) was provided for 10 weeks to the HFD group. Each mouse was administered 25 mg of THI orally, as determined in previous studies. For the vehicle treatment group, the same volume of distilled water was administered orally every day for 10 weeks by the same method used for THI treatment. Each group comprised six mice, and all mice were allowed free access to the described diet and water during experimental periods. Body weights and food intake were measured weekly at regular times.

Mice were dissected to collect tissues for analysis. Blood was collected from the retro-orbital sinus by using sodium-heparinized microhaematocrit capillary tubes (Marienfeld-superior, Lauda-Königshofen, Germany) and then transferred to Eppendorf tubes and incubated at room temperature. After 3 hr, all samples were centrifuged at 13,500 rpm for 5 min, after which serum was taken to measure triglyceride, glucose, and total cholesterol levels using an automatic blood chemistry analyzer Dry-Chem 4000i (Fujifilm, Saitama, Japan) at the Yonsei Laboratory Animal Research Center.

For exercise experiments, exercise groups of HFD mice were divided into two groups. One group was subjected to exercise without administration of THI while the other group was subjected to swimming exercise with oral administration of 25 mg of THI to each mouse. Mice underwent compulsory swimming exercise at 9:00 am and 5:00 pm twice a day, 5 days per week for 10 weeks. In the first week, mice swam voluntarily for 40 min, and this time was increased gradually up to 60 min for intensification of exercise. Mice swam at 31℃ in a temperature-controlled water bath, and the water bath was cleaned once a week.

Total RNA samples were prepared by homogenizing epididymal tissues and liver tissues with 500 µL of RNA iso-plus (Takara, Shiga, Japan). Prepared total RNAs were reverse-transcribed using M-MuLV reverse transcriptase (Promega, Madison, WI, USA) at 37℃ for 1 hr. PCR for amplification of mRNAs encoding hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL), mitochondrial uncoupling protein 2 (UCP2), and carnitine palmitoyltransferase 1a (CPT1a) was performed using appropriate primer pairs: ATGL forward, 5'-CTCCGAGAGATG TGCAAACA-3', reverse, 5'-CAGTTCCACCTGCTCAGACA-3'; HSL forward, 5'-CTTCCTGCAAGAGTATGTCACG-3', reverse, 5'-TGGAGGTGAGATGGTGACTG-3; UCP2 forward, 5'-CTGGCAGGTAGCACCACAGGTG-3', reverse, 5'-GCATGGTAAGGGCACAGTGAC-3'; CPT1a forward, 5'-GTCTGGAATCAACTCCTGGAAG-3', reverse, 5'-CAGTGACGTTGGAAGCTGTAG-3'. RT-PCR products were visualized by 1% agarose gel electrophoresis, and the intensity of the bands was measured using a DNR Bio-Imaging system (Kiryat Anavim, Jerusalem, Israel).

Mice were fasted for 14 hr before experiments, and D-glucose (Duchefa Biochemie, Haarlem, Netherlands) was administered to mice at a dose of 1 g per kg of body weight. Glucose level of the blood taken from the tail vein was measured using Accu-Chek (Roche Diagnostics, Basel, Switzerland) at 30 min intervals for 120 min.

The 3T3-L1 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Two days after reaching confluence (day 0), 3T3-L1 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 1 µg/mL of insulin, 0.25 µM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 10% fetal bovine serum (differentiation induction medium) for 2 days. Cells were then maintained in DMEM containing 1 µg/mL of insulin and 10% fetal bovine serum (differentiation maintenance medium). The differentiation maintenance medium was changed every 2 days until the cells were harvested on day 7. To test the effects of THI on adipogenesis, various concentrations of THI were added to the differentiation induction and maintenance media until cells were harvested. Differentiated cells in each well of 6-well plates were harvested in 500 µL of phosphate-buffered saline (PBS), frozen, and then sonicated. The triglyceride contents of the cell lysates were measured using a TG-S reaction kit (Asan Pharm., Seoul, Korea). Lipid droplets in cells were stained with Oil Red O, as previously described [13].

Cultured and differentiated cells were harvested using a cell scraper and lysed with ice-cold RIPA buffer containing 25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, and a protease inhibitor cocktail (Sigma-Aldrich). The total cell lysates were centrifuged at 14,000 rpm for 20 min at 4℃ to remove insoluble materials. The protein concentrations were determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). Fifty micrograms of protein extract was resolved by 10% SDS-polyacrylamide gel electrophoresis at 150 mA for 1 hr and then transferred to nitrocellulose membranes. The membranes were next blocked for 2 hr at room temperature with PBS containing 5% skim milk and 0.1% Tween 20, incubated with 1:1,000-dilutions of primary antibodies overnight at 4℃, and then with horseradish peroxidase-conjugated anti-rabbit secondary antibody for 1 hr at room temperature. Peroxidase activity was visualized using an ECL kit (Pierce, Rockford, IL, USA).

Statistical analysis was performed using SPSS statistical software. When differences among the groups were detected by one-way factorial ANOVA, Turkey's test was used. A level of significance of P < 0.05 was chosen for all statistical comparisons. Data are presented as means ± SEM.

Many herbal extracts were screened for the inhibition of fat accumulation in 3T3-L1 cells (data not shown). Two herbs, Scutellariae Radix and Platycodi Radix, were finally selected due to their strong activities. In this study, we examined the effects of the mixed extract of these two herbs, named THI, on metabolic abnormality in mice fed a high-fat diet. There are two types of obesity animal models available. One includes genetic models such as the leptin-deficient ob/ob mouse model and the leptin receptor deficient db/db mouse model. The other type includes the DIO model. In this study, we used the DIO model to determine the effects of THI on obesity since DIO is an experimental model with much higher applicability to human obesity. Mice were subdivided into three groups: the normal diet group with vehicle treatment, the 60% HFD group with vehicle treatment, and the 60% HFD group with THI treatment. There were significant differences in body weight between the HFD and normal diet groups from the first week of the experiment. The THI-administered mice showed a more modest increase in size and body weight than did the HFD group, although food intake was the same for both groups (Fig. 1A and 1B).

Weight of epididymal white adipose tissue was highly increased in the HFD group compared with the normal diet group, and the THI-administered HFD group showed a significant decrease in adipose tissue weight by as much as 40% compared with the HFD group (Fig. 2A). However, liver tissue weights were not significantly different among the three groups (Fig. 2B).

Since THI administration reduced adipose tissue weight, we wondered whether or not THI could induce changes in the serum levels of triglycerides and total cholesterol. The level of total cholesterol was lower compared with the HFD group (Fig. 3A and 3B), whereas the triglyceride level was not reduced to a statistically significant level. Next, to determine whether or not administration of THI enhances energy expenditure, total RNA was isolated from epididymal white adipose tissues. The mRNA levels of hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), which are involved in the mobilization of free fatty acids from adipose triacylglycerol stores, were analyzed by RT-PCR. There was no significant increase in HSL and ATGL mRNA expression (Fig. 3C). The mRNA levels of mitochondrial uncoupling protein 2 (UCP2) and carnitine palmitoyltransferase 1a (CPT1a), which are responsible for energy expenditure, were also analyzed by RT-PCR. The levels of UCP2 and CPT1a mRNA in adipose and muscle tissues were not changed statistically (Fig. 3E and 3F).

THI was also found to inhibit Oil red O-stained fat droplet formation, which is a marker of adipogenesis, during adipogenic differentiation of 3T3-L1 cells in vitro (Fig. 4A). THI also significantly reduced the intracellular fat content in a dose-dependent manner (Fig. 4B). To elucidate the molecular mechanism of THI action in vitro, the expression levels of major transcription factors of adipogenesis were analyzed. It was found that THI downregulated the expression of C/EBPα and PPARγ, whereas C/EBPβ levels were not changed (Fig. 4C).

Next, we performed an oral glucose tolerance test. All groups of mice were fasted during the night cycle, and glucose was orally injected 14 hr after starvation. Blood glucose levels were measured at the indicated time points. The THI-administered group showed improved oral glucose tolerance compared with the HFD group, although the glucose tolerance of the THI-administered group was not improved to the levels of the normal diet group (Fig. 5).

Since it is well-known that exercise effectively improves the metabolic index, we examined whether or not THI administration could act synergistically with exercise. The HFD group was orally administered with THI and subjected to swimming exercise for 10 weeks or swimming exercise without THI administration. Exercise induced dramatic weight loss in the THI-administered high-fat group (Fig. 6A) but did not alter the amount of food intake (Fig. 6B). Weight loss in the THI-administered exercised group was due to the dramatic reduction of adipose tissue (Fig. 6C) and triglyceride levels (Fig. 6D). The simultaneous combination of exercise and THI administration induced lower levels of total cholesterol compared with the group that only exercised (Fig. 6E). These results indicate that the simultaneous combination of exercise and THI administration has a synergistic effect on the reduction of total cholesterol levels and that THI does not affect the beneficial effect of exercise. In addition, the results also confirm an improvement in fasting blood glucose levels and oral glucose tolerance (Fig. 6F).