TRIzol reagent and SuperScript III kit were obtained from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Mouse primary antibodies against PPARγ (sc-7273), C/EBPα (sc-166258) and β-actin (sc-47778), and goat-anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies (sc-2030) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Primary antibodies were diluted to 1:1,000 and secondary antibodies at 1:5,000. Garcinia cambogia (GC) extract was purchased from Prakruti Products Pvt. Ltd. (Karnataka, India).

E. supina was obtained from Gungangbogam (Jechon, Korea). The dried and powdered fruit of E. supina (500 g) were extracted using 50% ethanol for 2 h under mantle-reflux at 80°C. Following filtration, and solvent removal on a rotary vacuum evaporator (N-000; EYELA; Tokyo Rikakikai Co., Ltd., Tokyo, Japan) ESEE was obtained as a powder (17). The yield of ESEE obtained using this procedure was 83.5 g (16.7% wt/wt). ESEE was stored at 4°C until required.

Male C57BL/6 mice (n=36; weight, 20.4±1.03 g; age, 5 weeks) were purchased from Samtako Bio Korea (Samtako Bio Korea, Osan, Korea). Experimental procedures were conducted according to a protocol approved by the institutional animal care committee of Chonbuk National University. Mice were housed at 22±2°C, 50±5% humidity and provided a normal diet and water ad libitum. Following a 1-week acclimation period, mice were divided into six groups (n=6 each), as follows: i) the normal diet (ND) group, fed with normal standard diet containing 14% fat, 21% protein and 65% carbohydrate (5L79; Orient Bio, Inc., Seongnam, Korea); ii) the high-fat diet (HFD) group, fed with HFD containing 60% fat, 20% protein and 20% carbohydrate (D12492; Research Diets, Inc., New Brunswick, NJ, USA); iii) the HFD+ESEE (2) group, fed with HFD and treated with ESEE (2 mg/kg/daily); iv) the HFD+ESEE (10) group, fed with HFD and treated with ESEE (10 mg/kg/daily); v) the HFD+ESEE (50) group, fed with HFD and treated with ESEE (50 mg/kg/daily); vi) the HFD+GC group, fed with HFD and treated with GC (200 mg/kg/daily). ESEE or GC was administered daily via oral gavage for 6 weeks. Body weights and amounts of food consumed were determined weekly.

Following the 6-week treatment period, mice were starved for 6 h prior to sacrifice and anesthetized via intraperitoneal injection of ketamine (75 mg/kg; Yuhan Corporation, Seoul, Korea) and rompun (15 mg/kg; Bayer Korea Ltd., Seoul, Korea). Images were acquired using a Skyscan-1076 micro-CT scanner (Bruker microCT, Kontich, Belgium). CT was performed using a pixel size of 35 μm, a source voltage of 50 kVp, and a source current of 200 μA. The X-ray detector contained a 12-bit, water-cooled charge-coupled device camera with a resolution of 4,000×2,300 pixels and a scintillator. Images were acquired at 0.6 degrees and an exposure time of 0.46 sec using a 1-mm aluminum energy filter. Mice were sacrificed following micro-CT scanning and abdominal fat volumes were measured using an Olympus SP-500 UZ camera (Olympus Corporation, Tokyo, Japan). Following micro-CT, under anesthesia, mice were euthanized via intracardiac puncture. Blood was collected (0.8-0.9 ml) via cardiac puncture followed by removal of organs such as liver, kidney and spleen. Following exsanguination, mortality was confirmed by incising the heart and ensuring that no respiratory movement occurred for ≥3 min.

Liver tissue and epididymal white adipose tissue (eWAT) were fixed using 10% neutral buffered formalin at room temperature for 8 h, embedded in paraffin wax, and cut serially into 10-μm sections. Sections were stained with H&E at room temperature (hematoxylin, 4 min; eosin, 2 min) and histologic alterations were observed and photographed under a light microscope (magnification, ×100; Olympus CX21; Olympus Corporation) (18).

To evaluate hepatic steatosis, levels of hepatic total triglycerides (TG) and cholesterol (TC) were determined by homogenizing liver tissues in a chloroform/methanol mixture (2:1, v/v). TG and TC concentrations were then measured using commercially available kits (TG; AM 1575-K; TC; AM 202-K; Asan Pharmaceutical Co., Ltd., Seoul, Korea) as detailed previously (19). In addition, rapid enzymatic assay kits were used to measure serum levels of TC, TG and high-density lipoprotein cholesterol (HDL-c; AM 203-K; Asia Pharmaceutical Co., Ltd., Seoul, Korea), and low density lipoprotein cholesterol (LDL-c) was calculated using Friedewald's formula (20) as follows: LDL-c=TC-(HDL-c+TG/5).

ELISA kits were used to measure leptin (ADI-900-019A; Enzo Life Sciences Inc., Farmingdale, NY, USA) and adiponectin (47-ADPMS-E01; R&D Systems, Inc., Minneapolis, MN, USA). HFD-induced liver damage was assessed by measuring the serum enzyme activities of alanine transaminase (ALT) and aspartate aminotransferase (AST) using the ALT/AST cassette test kit (Alere Cholestech LDX^® System; Alere, Inc., Waltham, MA, USA) (18).

Total RNA was extracted from eWAT and liver tissues using TRIzol RNA Isolation Reagent, according to the manufacturer's instructions. The synthesis of cDNA was performed with 2 μg RNA using the SuperScript III First Strand Synthesis kit protocol. qPCR was performed using the ABI Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using SYBR Green PCR Master Mix (Thermo Fisher Scientific, Inc.). PCR was performed using the following conditions: 95°C for 10 min followed by 40 cycles of denaturation at 95°C for 15 sec, and annealing at 60°C for 1 min, as detailed previously (21). Primer sequences were as follows: GAPDH, forward 5′-CATGGCCTTCCGTGTTC-3′ and reverse 5′-CCTGGTCCTCAGTGTAGC-3′; PPARγ, forward 5′-GATGGAAGACCACTCGCATT-3′) and reverse 5′-AACCATTGGGTCAGCTCTTG-3′; and C/EBPα, forward 5′-TTGTTTGGATTTATCTCGGC-3′ and reverse 5′-CCAAGAAGTCGGTGGACAAG-3′. The comparative quantification cycle method was used to measure the relative quantitation of each gene and mRNA levels were normalized with GAPDH, as detailed previously (6).

eWAT and liver tissues were lysed in ice-cold radioimmunoprecipitation assay buffer (sc-24948; Santa Cruz Biotechnology, Inc., CA, USA) for 40 min and centrifuged (12,000 × g) for 20 min at 4°C, as detailed previously (21). Protein quantification was determined via bicinchoninic acid assay and protein samples (20 μg/lane) were separated by 8% SDS-PAGE and transferred to polyvinylidene difluoride membranes (GE Healthcare, Chicago, IL, USA), which were then blocked with 5% skimmed milk in tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature. Membranes were then probed with primary antibodies at 4°C overnight, washed with TBST 4 times, incubated at room temperature with HRP-conjugated secondary antibody for 45 min, and rewashed with TBST 3 times. Proteins were visualized using an enhanced chemiluminescence detection kit (EMD Millipore, Bedford, MA, USA) and a Fusion FX7 imaging system (Vilber Lourmat, Marne-la-Vallée, France).

UPLC was performed using an ACQUITY UPLC BEH C18 column (2.1×50 mm, 1.7 μm) and a photodiode array detector (Waters Corporation, Milford, MA, USA), as detailed previously (17). Elution was performed at a flow rate of 0.15 ml/min using distilled water containing 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent B) in gradient mode (B 5% from 0-1 min, B 5-95% from 1-16 min, B 95-100% from 16-18 min, and B 100% from 18-26 min at a flow rate of 0.15 ml/min at 25°C. Detection was performed at a wavelength of 330 nm.

Data are presented as the mean + or ± standard error of the mean, and were analyzed using GraphPad Prism software (version 5.0; GraphPad Software, Inc., La Jolla, CA, USA). One-way analysis of variance was used to measure the significant difference followed by Tukey's post hoc test for comparison of means. P<0.05 was considered to indicate a statistically significant difference.

To investigate the anti-obesity effect of ESEE, mice were fed HFD with or without ESEE (2, 10 and 50 mg/kg, oral gavage daily) or with GC (200 mg/kg, oral gavage daily) for 6 weeks. Food intake and body weights were recorded once weekly. At the end of the 6-week treated period, HFD mice had a mean body weight gain of 41.2%, whereas ND mice exhibited a gain of 18.7%. Mean percentage body weight gain of HFD mice was significantly reduced by ESEE (50 mg/kg) supplementation (Fig. 1A and B). In addition, no significant difference in dietary intake was observed among the HFD-fed and ESEE administered groups (Fig. 1C), but 50 mg/kg ESEE significantly reduced the food conversion efficiency as compared with HFD-fed mice (Fig. 1D). These results indicate that ESEE induced adipose weight loss, and reduced body weight gain and the percentage of food efficiency.

Micro-CT analysis results demonstrated that orbital fat volume percentage was significantly lower in ESEE (10 and 50 mg/kg) treated mice than in HFD-fed mice (Fig. 2A and B). Furthermore, the effects of ESEE supplementation on fat accumulation in eWAT, liver, spleen and kidneys were investigated. The results demonstrated that these organs were significantly heavier in HFD mice than in ND mice, and that ESEE treatment significantly reduced fat deposition in these organs, compared with HFD-fed mice (Fig. 2C–G).

Increases in lipid accumulations in the liver and eWAT are commonly observed in HFD-fed obese mice (17). H&E staining revealed enlarged or hyper-trophic eWAT in the HFD group, and smaller eWAT sizes in ESEE supplemented mice (Fig. 3A). In addition, the histological study revealed enlarged hepatocytes, as evidenced by excessive vacuolation in the HFD group as compared with the ND group. As presented in Fig. 3B, ESEE markedly reduced vacuolization and lipid droplet numbers in the liver tissues of HFD mice. Hepatic steatosis was determined by measuring hepatic TG and TC levels. The present results demonstrated that ESEE (10 and 50 mg/kg) supplementation significantly reduced HFD-induced levels of TG and TC in liver tissues (Fig. 3C and D). Serum ALT and AST levels were also measured, which are clinical markers of liver damage. The results indicated that ESEE significantly reduced the HFD-induced serum levels of AST and ALT compared with the HFD group (Fig. 3E and F). These data indicate that ESEE inhibited HFD-induced hepatic steatosis and serum liver enzymes.

HFD-fed mice exhibited significantly higher serum levels of TC, low-density lipoprotein cholesterol (LDL-c) and TG, and significantly lower levels of high-density lipoprotein cholesterol (HDL-c) than mice in the ND group. ESEE significantly inhibited TC (50 mg/kg), LDL-c (10 and 50 mg/kg) and TG (50 mg.kg) increases, and increased HDL-c levels (10 and 50 mg/kg) vs. HFD-fed mice (Fig. 4A–D). Furthermore, ESEE significantly inhibited leptin levels and increased adiponectin levels. At a dose of 50 mg/kg, ESEE decreased mean leptin levels by 64.7% (Fig. 4E) and increased mean adiponectin levels by 51.1% compared with the HFD group (Fig. 4F).

RT-qPCR and western blotting were performed to measure the mRNA and protein expressions, respectively, of transcription factors of adipogenesis, such as PPARγ and C/EBPα. The results demonstrated that ESEE (10 and 50 mg/kg) significantly inhibited the mRNA and protein expressions of PPARγ and C/EBPα in eWAT tissues (Fig. 5) and liver tissues (Fig. 6), as compared with the HFD group.

UPLC assay demonstrated that corilagin and ellagic acid were the main components of ESEE (Fig. 7). However, other peaks observed were unidentified and require further study.