**1. Introduction**

Obesity is defined as excessive weight gain, particularly inordinate fat accumulation in the body [1]. Due to rapid economic growth and westernized diets, the number of patients with obesity has increased [2], resulting in increased rates of diseases such as type 2 diabetes [3], hypertension [4], hypercholesterolemia [5], and hyperlipidemia [6].

Obesity occurs because of a combination of overeating, lack of exercise, and neurotransmitters, drugs, and genetic factors [7]. To improve obesity, pharmacological treatment has been applied. However, because these treatments involve diverse side effects such as diarrhea and vomiting, the development of anti-obesity materials from natural products with few side effects is required [8,9].

*Nelumbo nucifera* L contains several flavonoids and alkaloids and has recently been used as a plain or blended tea to treat obesity in China [10]. *Morus alba* L is abundant in polyphenols such as rutin and quercetin and has been used to treat dyslipidemia, diabetes, fatty liver disease, and hypertension [11]. *Raphanus sativus* is an annual herb belonging to the cruciferous family and has been reported to be effective for treating hyperlipidemia by decreasing blood sugar level [12]. Some researchers evaluated the anti-obesity effects of *Eriobotrya japonica* and *N. nucifera* in adipocytes and obese mice using a single material or mixture, but few studies have examined mixtures of three natural materials [13]. Because each material has a different physiological activity, it is necessary to confirm the physiological activities of each according to the type and mixing ratio. These materials may exert synergistic effects, but there might be also negative effects [14,15].

In this study, we blended *N. nucifera* L., *M. alba* L., and *R. sativus* in different mixing ratios and selected the optimal mixed material based on antioxidant and anti-adipogenic experiments. The anti-obesity effects of optimal mixed material were evaluated in 3T3-L1 adipocytes and C57BL/6J obese mice.

#### **2. Materials and Methods**

## *2.1. Sample Preparation*

*Nelumbo nucifera* leaves, *M. alba* leaves, and Dried *R. sativus* root were supplied by Haram Co. Ltd. (Jeungpyeong, Korea) and freeze-dried and mixed in 13 ratios. The mixing ratio of *N. nucifera* L, *M. alba* L, *R. sativus* is 80:20:0 (M1), 70:30:0 (M2), 60:40:0 (M3), 50:50:0 (M4), 80:0:20 (M5), 70:0:30 (M6), 70:20:10 (M7), 60:30:10 (M8), 60:20:20 (M9), 50:30:20 (M10), 100:0:0 (M11), 0:100:0 (M12), 0:0:100 (M13). These materials were extracted by boiling and reflux with hot water (WM) and 70% ethanol (EM) at 60 ◦C for 12 h. Then they were filtered, concentrated by a vacuum evaporator (Rotavapor R-200; Buchi, Flawil, Switzerland), and freeze-dried (Bondiro; Il Shin Lab Co. Ltd., Seoul, Korea) to obtain a powder [16].

#### *2.2. Antioxidant Activity Analysis*

We performed a DPPH (<sup>α</sup>,<sup>α</sup>-Diphenyl-β-picrylhydrazyl) assay as described previously with some modifications [17]. First, 0.8 mL of 0.4 mM DPPH solution was added to 0.2 mL of the sample, and the mixture was incubated in the dark for 10 min. Next, it was measured at the 517 nm absorbance using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

$$\text{DPPH} \text{ radical scavenging activity (\%)} = (1 - (\text{A}\_{\text{experiment}} / \text{A}\_{\text{control}})) \times 100 \tag{1}$$

Aexperiment: Absorbance of experimental group, Acontrol: Absorbance of control group.

The total phenolic contents were determined using Folin-Ciocalteu's colorimetric method [18]. First, 1 mL of sample was added to 10% Folin-Ciocalteu reagen<sup>t</sup> and 2% Na2CO3 reagen<sup>t</sup> in order and incubated for 1 h in the dark. It was measured at the 750 nm absorbance using a microplate reader. Gallic acid was used as a standard and the total phenolic contents were calculated from the standard calibration curve (*y* = 19.12*x* − 0.0261, *R*<sup>2</sup> = 0.9992).

#### *2.3. Cell Culture and Di*ff*erentiation*

3T3-L1 preadipocytes were obtained from American Type Culture Collection (CL-173, ATCC, Manassas, VA, USA) and grown in culture plates containing Dulbecco's modified Eagle's medium (Lonza, Basel, Switzerland) supplemented with 10% bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (P/S; Gibco) kept at 37 ◦C and 5% CO2 incubator condition. 3T3-L1 preadipocytes were differentiated to adipocytes at the 2 day after confluence by exchanging with MDI medium (DMEM containing 10% fetal bovine serum (Gibco), 1% P/S, 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich, Saint Louis, MO, USA), 1 μM dexamethasone (Sigma-Aldrich), and 1 μg/mL insulin (Gibco)). Every 2 days during incubation, the culture medium was changed to DMEM containing 10% fetal bovine serum and 1 μg/mL insulin with extracts until day 6 [19].

#### *2.4. Cell Viability Assay*

3T3-L1 preadipocytes were plated into 96-well plates (1 × 10<sup>6</sup> cells/well) and differentiated with MDI medium along with 100 μg/mL extracts since the 2 day after confluence for 6 days. At the day 6, Mixed XTT (2,3-bis(2-methoxy-4-nitro-5- sulfophenyl)-2*H*-tetrazolium-5-carboxanilide) and PMS (*N*-methyl dibenzopyrazine methyl sulfate reagents) (WelGene, Seoul, Korea) were included into the medium and incubated for 4 h at 37 ◦C. Then the soluble formazan salt generated in the medium was measured at 450 nm against 690 nm using a microplate reader [20].

#### *2.5. Oil-Red O Staining Assay*

The amount of lipid accumulation of 3T3-L1 cells differentiated on a 24-well plate for 6 days was determined using Oil-Red O staining. In brief, the cells were washed with phosphate-buffered saline (PBS; Gibco) and fixed in 10% formaldehyde in distilled water for 1 h. Next, the cells were dried with 60% isopropanol and stained with Oil-Red O (Sigma-Aldrich) solution for 1 h, and then washed with distilled water. The stained lipids were eluted with 100% isopropanol and measured using a microplate reader at 490 nm.

#### *2.6. Animal Experiment Design*

Eight-week-old male C57BL/6J mice were obtained from Korea BioLink (Eumseong, Korea) and maintained under standard light (12:12-h light/dark cycle), temperature (22 ± 2 ◦C) and humidity (55 ± 15%) conditions. The diets included a normal diet (AIN-76A; Research Diets, Inc., New Brunswick, NJ, USA) and high-fat diet (HFD; D12492; Research Diets, Inc.), and obesity was induced in the mice fed an HFD for 2 weeks. After 2 weeks, the mice were randomly divided into eight groups; C57BL/6J normal group, 60% kcal HFD control group, positive control group (*Garcinia cambogia*, GC\_245 mg/kg), EM11\_100 mg/kg group, EM12\_100 mg/kg group, EM01\_50 mg/kg group, EM01\_100 mg/kg group, quercetin-3-O-glucuronide (Q3OG) 10 mg/kg group; *n* = 10). Meanwhile, *Garcinia cambogia* is an ingredient in dietary supplements for weight loss [19]. These test substances were taken orally with saline solution once a day for 8 weeks. The mice had free access to food and water, and their body weight and calorie intake were measured weekly. Biochemical measurements were analyzed after treatment of the candidate materials for 8 weeks and glucose tolerance test were performed 3 days later. Experiments were carried out with the approval of the Institutional Animal Care and Use Committee of Kangwon University for the ethical and scientific feasibility study and effective managemen<sup>t</sup> of animal experiments (Permit No. KIACUC-12-0140).

#### *2.7. Glucose Tolerance Test*

After administering the anti-obesity candidate materials for 8 weeks, mice were fasted for 12 h and the blood glucose level was measured at 0, 15, 30, 45, 60, and 75 min after glucose (1 g/kg) peritoneal administration. Blood for glucose measurement was obtained from the tail vein of mice and measured using a serum analyzer (Accutrend plus GCTL Cobas Roche, Basel, Switzerland).

#### *2.8. Serum Biochemical Parameter Analysis*

After administering the anti-obesity candidate materials for 8 weeks, blood samples were collected by cardiac puncture using centrifugation (2000× *g*, 4 ◦C, 15 min) and stored at −74 ◦C. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine, which are indices of liver and kidney function, and total cholesterol (TC), high-density lipoprotein cholesterol (HDL-cholesterol), low-density lipoprotein cholesterol (LDL-cholesterol), triglyceride (TG), non-esterified fatty acid (NEFA), and glucose, which are indices of lipid content, were measured using a biochemical automatic analyzer (Hitachi-720, Hitachi Medical, Tokyo, Japan).

#### *2.9. Serum Adipokine Analysis*

Also, adiponectin, leptin, and insulin-like growth factor-1 (IGF-1) were isolated in blood samples collected by cardiac puncture after administration of anti-obesity candidate materials for 8 weeks. Each antibody was diluted in coating buffer and coated on a microwell for overnight incubation at 4 ◦C. Each well was washed three times with washing buffer, and 100 μL of serum was dispensed, incubated at room temperature for 1 h, and washed twice with washing buffer. Thereafter, 100 μL of the antibody avidin-horseradish peroxidase conjugate was added and incubated at room temperature for 1 h, and then washed again. The TMB substrate was dispensed in 100 μL, incubated in the dark for 30 min, and treated with 50 μL of stop solution, after which absorbance was measured at 450 nm.

#### *2.10. Real-Time Polymerase Chain Reaction (RT-PCR)*

Total cellular RNA was extracted from the liver, epididymal, and abdominal subcutaneous adipose tissue using a homogenizer and Trizol reagen<sup>t</sup> (Sigma-Aldrich). Total RNA was used for cDNA synthesis with the One-step SYBR Green PCR kit (AB Science, Avenue George V, France). The Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) used for real-time quantitative PCR. The probes containing the fluorescence reporter dye 6-carboxy-fluorescein (Applied Biosystems) was used to indicate mRNA gene expression. The mouse glyceraldehyde-3-phosphate dehydrogenase probe (VIC/MGB probe, primer limited, 4352339E, Applied Biosystems) was used as an internal standard. The sequence of the used primer was as follows; Leptin sense (5-AACCCTTACTGAACTCAGATTGTTAG-3) and antisense (5-TAAGTCAGTTTAAATGCTTAGGG-3); PPARγ sense (5-ATGCCATTCTGGCCCACCAACTT-3) and antisense (5-CCCTTGCATCCTTCACAAGCATG-3); PPARα sense (5-GCCTGTCTGTCGGATGT-3) and antisense (5-GGCTTCGTGGATTCTCTTG-3); Adiponectin sense (5-TTCAAATGAGATTGTGGGAAAAT-3) and antisense (5-ACCGATACAGTACAGTACAGTA-3); UCP1 sense (5-CGACTCAGTCCAAGAGTACTTCTCTTC-3) and antisense (5-GCCGGCTGAGATCTTGTTTC-3); UCP2 sense (5-TTCAAATGAGATTGTGGGAAAAT-3) and antisense (5-ACCGATACAGTACAGTACAGTA-3); ACX1 sense (5-CAGGAAGAGCAAGGAAGTGG-3) and antisense (5-CCTTTCTGGCTGATCCCATA-3); DGAT1 sense (5-TGCTACGACGAGTTCTTGAG-3) and antisense (5-CTCTGCCACAGCATTGAGAC-3); SCD1 sense (5-CATCGCCTGCTCTACCCTTT-3) and antisense (5-GAACTGCGCTTGGAAACCTG-3); SREBP1c/ADD1 sense (5-AGCCTGGCCATCTGTGAGAA-3) and antisense (5-CAGACTGGTACGGGCCACAA-3); ACS1 sense (5-TCCTACAAAGAGGTGGCAGAACT-3) and antisense (5-GGCTTGAACCCCTTCTGGAT-3); CPT1b sense (5-GTCGCTTCTTCAAGGTCTGG-3) and antisense (5-AAGAAAGCAGCACGTTCGAT-3); FAS sense (5-CTGAGATCCCAGCACTTCTTGA-3) and antisense (5-GCCTCCGAAGCCAAATGAG-3); GAPDH VIC (5-TGCATCCTGCACCACCAACTGCTTAG-3). The PCR conditions were 50 ◦C for 2 min, 94 ◦C for 10 min, 95 ◦C for 15 s, and 45 ◦C for 1 min of 40 cycles.
