## 2 Materials and Methods
### Experimental Animals  
The male C57 BL/6] mice (purchased from Central Lab. Animal Inc., Seoul, Korea) were housed and maintained under a pathogen-free environment in controlled conditions (12 h light cycle, room temperature: 24 \(\pm\) 1 \({}^{\circ}\)C, and relative humidity: 50-60%). Mice had free access to a standard chow diet and water for 1 week for adaptation. The Wonkwang University Animal Ethics Committee approved the animal experiment protocols (Approval No.: WKU19-78).  
After 1-week of adaptation, mice (age 4 weeks) were randomly divided into four groups (n = 6). A normal diet group was fed with the standard chow diet (normal diet: 5L79 Orient Bio Inc., Seongnam, Korea) consisting of 65.30% carbohydrate, 20.1% protein, and 13.67% fat for 13 weeks. The remaining three groups were fed with a high-fat diet (HFD: Rodent Diet D12451, Research Diets, New Brunswick, NJ, USA) consisting of 35% carbohydrate, 20% protein, and 45% fat. To induce obesity in mice, only HFD was fed for 5 weeks without drug treatment. In 5 weeks, the mice fed with the HFD were considered to induce obesity (as the average body weights of HFD-fed mice was more than 19% higher than the standard diet-fed mice). Thereafter, the DIO mice were treated with the respective drugs or vehicle for another 7 weeks with continued feeding of an HFD. Among three HFD-fed groups, one group was treated with vehicle (0.2% CMC/PBS) orally, considering as an HFD control. The standard diet-fed group was also treated with vehicle. The second HFD-fed group was treated with formulation F2 in a dose of 46 mg/kg, p.o. two times a day (F2-46). By analysing the effectiveness of two used doses in the previous study [22], a single higher dose of F2 (46 mg/kg) was used in this study. The third HFD-fed group was used as a positive control and treated with _Garcinia cambogia_ (GC) in a dose of 200 mg/kg [24], twice daily. Weekly recordings of the body weight and food intake were conducted. Food efficiency ratios (FER) were calculated. At the end of the treatment period, the mice had fasted for 14 to 15 h and blood samples were collected from the eyes, and mice were sacrificed by cervical dislocation. After sacrifice, epididymal white adipose tissue (WAT), liver, kidney, and spleen were isolated and weighed. Liver and WAT were immediately fixed into 10% formalin for histological analysis. Some portions of the adipose tissue were stored at -70 \({}^{\circ}\)C for real-time PCR analysis. Fasting blood glucose was measured from the tail blood before sacrifice by using a one-touch glucometer (CareSens \({}^{\circledR}\)N, i-sens, Seoul, Korea).  
The serum samples were prepared by centrifugation of the collected blood samples (14,000 rpm for 20 min at 4 \({}^{\circ}\)C) and then stored at -70 \({}^{\circ}\)C for biochemical analysis. The serum levels of triglyceride and total cholesterol were determined using respective assay kits (BioVision, Milpitas, CA, USA) according to the manufacturer's procedure. The serum insulin was determined by using the Mouse Insulin ELISA Kit (Cat#638-01489, Shibayagi Co. Ltd., Gunma, Japan) according to the manufacturer's procedure. The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as follows [25].  
HOMA-IR = [Fasting insulin (\(\mu\)U/mL) \(\times\) fasting glucose (mmol/L)]/22.5.  
The histological analyses of tissues (liver and WAT) were conducted by haematoxylin and eosin (H&E) staining using the same method described in the previous report [22].