**3. Results**

#### *3.1. E*ff*ects of 26 Extracts by Mixture Ratio of N. Nucifera L., M. alba L., R. Sativus on the Antioxidant and Anti-Adipogenic Activities*

<sup>α</sup>,<sup>α</sup>-Diphenyl-β-picrylhydrazyl (DPPH) is a very stable free radical and representative reaction substance used to measure antioxidant capacity. DPPH radical scavenging activity assay is a method that utilizes the principle that a purple compound is discolored as yellow when radicals are eliminated through hydrogen donation in a phenolic compound or flavonoid with a hydroxyl radical (-OH) [21]. For the extraction method, the radical scavenging activities of ethanol extracts were superior to those of hot water extracts. For the mixing ratio, EM05 (84.30%), EM06 (83.00%), and EM01 (81.65%) were superior in order, but there was no significant difference between these samples (Figure 1A).

(A) 

**Figure 1.** *Cont*.

(D) 

**Figure 1.** Effects of 26 extracts by mixture ratio of *Nelumbo nucifera* L., *Morus alba* L., and *Raphanus sativus* on antioxidant activity, cell viability, lipid accumulation. (**A**) DPPH radical scavenging activity. (**B**) Total phenolic contents. (**C**) Post-confluent 3T3-L1 preadipocytes were differentiated along with the treatment of each extracts for 6 days. XTT (2,3-bis(2-methoxy-4-nitro-5- sulfophenyl)-2*H*-tetrazolium-5-carboxanilide) and PMS (*N*-methyl dibenzopyrazine methyl sulfate reagents) mixture was added to the medium. After 4 h of incubation, cell viability was found out by calculating the absorbance at 450 nm against 690 nm. (**D**) Stained lipids were extracted and quantified by calculating the absorbance at 490 nm. Data are presented as the mean ± SEM (*n* = 3). Means with different letters on bars indicate that there is a significant difference at *p* < 0.05 by Duncan's multiple range test.

The content of phenol, a representative antioxidant, was highest in EM03 (49.00 mg GAE/g), EM02 (48.40 mg GAE/g), and EM01 (47.90 mg GAE/g), with no significant difference between these samples (Figure 1B).

No cytotoxicity or changes in morphology were observed at the concentration of 100 μg/mL mixtures by XTT assay (Figure 1C). Next, the anti-adipogenic activity was measured by Oil-Red O staining, and EM01 (75.30%) was found to be the most e ffective mixture for inhibiting lipid accumulation among the 26 mixtures (Figure 1D). Therefore, we selected EM01 as an optimal mixture and carried out anti-obesity experiments using in vitro and in vivo models.

#### *3.2. E*ff*ect of EM01 on Lipid Accumulation*

We previously found that quercetin-3-O-glucuronide is a bioactive compound in mixed materials [16]. In antioxidant and anti-obesity experiments, EM01 (100 μg/mL) was measured to determine its anti-obesity activity with quercetin-3-O-glucuronide (7.8 μM). As shown in Figure 2, EM01 (80.73%) showed better lipid accumulation inhibitory activity than the single materials, EM11 (84.92%), EM12 (86.39%), and single bioactive compound treatment (84.65%). There may have been a synergistic interaction in EM01 between quercetin-3-O-glucuronide and other compounds.

**Figure 2.** Effect of EM01 and its bioactive compound on lipid accumulation. Post-confluent 3T3-L1 preadipocytes were di fferentiated along with the treatment of each extracts and its bioactive compound for 6 days. Stained lipids were eluted and quantified by calculating the absorbance at 490 nm. Data are presented as the mean ± SEM (*n* = 3). Means with di fferent letters on bars indicate that there is a significant di fference at *p* < 0.05 by Duncan's multiple range test.

#### *3.3. E*ff*ects of EM01 on Body Weight, Food Intake, FER, Organ Weight, and Adipose Tissue Weight in HFD-Induced Obese Mice*

There was no significant di fference in the initial body weight between experimental groups, but the final body weights in di fferent groups were significantly di fferent. The body weight increased significantly in the HFD-CTL group compared to in the ND group, suggesting that obesity was induced by the HFD. The HFD-EM01 group (100 μg/mL) showed a higher weight loss rate than the HFD-CTL group (Figure 3A). Food intake was not significantly di fferent between groups except for the ND group, and the food e fficiency ratio (FER) was significantly decreased in the HFD-EM01 group (100 μg/mL) (Figure 3B,C).

*Foods* **2019**, *8*, 170

**Figure 3.** Effects of EM01 on **(A)** body weight, **(B)** food intake, **(C)** FER, **(D)** organ weight, and **(E)** adipose tissue fat weight in high-fat diet (HFD)-induced obese mice. GC, *Garcinia cambogia* extract; HFD, mice were fed a high-fat diet (60% kcal fat); ND, mice were fed a normal diet (10% kcal fat); FER, food efficiency ratio (total weight gain/total food intake × 100). Data are presented as the mean ± SEM (*n* = 12); # *p* < 0.05, ## *p* < 0.01, ### *p* < 0.001 vs. ND; \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001 vs. HFD-CTL.

The kidney and spleen weights were not significantly different among groups (Figure 3D). Liver weight increased significantly in the HFD-CTL group compared to in the ND group, and weight decreased significantly in the HFD-EM01 and HFD-Q3OG groups compared to in the HFD-CTL group. According to Figure 3E, the weight of the kidney adipose tissue significantly increased in the HFD-CTL group compared to in the ND group, but no significant inhibitory effect was observed in any group. The weight of abdominal subcutaneous fat, epididymal, and intestine adipose tissue increased significantly in the HFD-CTL group compared to in the ND group, but there was an inhibitory effect on fat accumulation in the HFD-EM01 and HFD-Q3OG group compared to the positive control (HFD-GC group).

#### *3.4. E*ff*ects of EM01 on glucose tolerance in HFD-induced obese mice*

When glucose tolerance occurs, blood glucose levels do not rise despite glucose administration, which is common in obese patients [22]. To investigate the effect of EM01 administration on glucose-induced hyperglycemia, glucose was orally administered, and the glucose tolerance test was performed over time. Blood glucose levels in all groups increased after 30 min of glucose injection. After 60 min of glucose injection, blood glucose levels in EM01 and Q3OG administrated groups dropped markedly even close to ND-group. On the other hand, it was confirmed that blood glucose levels did not decrease rapidly in EM11 and EM12 administrated groups (single material) (Figure 4).

**Figure 4.** Effects of EM01 on glucose tolerance in HFD-induced obese mice. Data are presented as the mean ± SEM (*n* = 12); # *p* < 0.05, ## *p* < 0.01, ### *p* < 0.001 vs. ND; \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001 vs. HFD-CTL.

#### *3.5. E*ff*ects of EM01 on the Serum Lipid Profile in HFD-Induced Obese Mice*

The HFD-CTL group showed significant increases in all parameters of the serum lipid profile compared to the ND group. The TC level was significantly decreased in the HFD-EM01 and HFD-Q3OG groups compared to in the HFD-CTL group (Figure 5A). HDL-cholesterol was lowered by more than LDL-cholesterol in these groups (Figure 5B,C). TC may decrease when LDL-cholesterol levels, often referred to as bad hormones, are suppressed [23]. The TG level was higher in the HFD-CTL group than in the ND group, but there was no significant difference between all groups compared to HFD-CTL (Figure 5D). AST, ALT, and creatinine are used as liver toxicity marker [24] and their levels were significantly lowered by EM01 administration (Figure 5E,F). This suggests that the 8-week oral administration did not affect liver and kidney toxicity in obese mice.

**Figure 5.** Effects of EM01 on serum lipid profile in HFD-induced obese mice. (**A**) Total cholesterol. (**B**) HDL cholesterol. (**C**) LDL cholesterol. (**D**) Triglyceride. (**E**) Creatinine. (**F**) AST & ALT. Data are presented as the mean ± SEM (*n* = 12); # *p* < 0.05, ## *p* < 0.01, ### *p* < 0.001 vs. ND; \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001 vs. HFD-CTL. AST, aspartate aminotransferase; ALT, alanine aminotransferase.

#### *3.6. E*ff*ects of EM01 on the Energy Balancing Metabolism in HFD-Induced Obese Mice*

Adipokine is a hormone specifically secreted from adipose tissue that affects endocrine system function. We analyzed adiponectin, leptin, IGF-1, which plays an important role in normal growth and health maintenance, NEFA, and glucose, which is associated with energy homeostasis [25]. Adiponectin levels in the serum were significantly decreased in the HFD-CTL group compared to in the ND group, but there was a significant increase in the HFD-EM01 and HFD-Q3OG groups compared to in the HFD-CTL group (Figure 6A). Leptin, IGF-1, NEFA, and glucose levels in the serum were significantly increased in the HFD-CTL group compared to in the ND group, while HFD-EM01 and HFD-Q3OG group were significantly decreased compared to the HFD-CTL group (Figure 6B–E).

**Figure 6.** Effects of EM01 on the energy balancing metabolism in HFD-induced obese mice. (**A**) Adiponectin. (**B**) Leptin. ( **C**) IGF-1. ( **D**) NEFA. (**E**) Glucose. Data are presented as the mean ± SEM (*n* = 12); # *p* < 0.05, ## *p* < 0.01, ### *p* < 0.001 vs. ND; \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001 vs. HFD-CTL. IGF-1, insulin-like growth factor-1; NEFA, non-esterified fatty acid.

#### *3.7. E*ff*ects of EM01 mRNA Expression Level of Lipid Metabolism-Related Genes in HFD-Induced Obese Mice*

We analyzed the mRNA levels of adipogenesis, lipogenesis, and fatty acid oxidation-related genes in the liver, epididymal adipose tissue, and abdominal subcutaneous fat after 8 weeks of EM01 administration (Figure 7).

As shown in Figure 7A, the mRNA levels of FAS, DGAT1, SCD-1, leptin, SREBP1c, PPARγ in the liver were significantly increased in the HFD-CTL group but decreased in the HFD-EM01 and HFD-Q3OG groups. The mRNA levels of COX1, adiponectin, UCP2, and PPARα increased in the HFD-EM01 and HFD-Q3OG groups compared to in the HFD-CTL group.

As shown in Figure 7B, the mRNA levels of the FAS, leptin in the epididymal adipose tissue increased in the HFD-CTL group, but markedly decreased in HFD-EM01 and HFD-Q3OG groups. The mRNA levels of the ACS1, ACOX1, CPT1b, UCP2, adiponectin, PPARα increased in HFD-EM01 and HFD-Q3OG groups compared to those in the HFD-CTL group.

As shown in Figure 7C, the mRNA level of UCP1 in abdominal subcutaneous fat significantly increased in the HFD-EM01 and HFD-Q3OG groups compared to in the HFD-CTL group.

**Figure 7.** *Cont*.

**Figure 7.** *Cont*.

**Figure 7.** Effects of EM01 on mRNA expression level of lipid metabolism-related genes in HFD-induced obese mice. (**A**) Liver. (**B**) Epididymal adipose tissue. (**C**) Abdominal subcutaneous fat. Data are presented as the mean ± SEM (*n* = 12); # *p* < 0.05, ## *p* < 0.01, ### *p* < 0.001 vs. ND; \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001 vs. HFD-CTL. FAS, fatty acid synthase; SCD-1, stearoyl-CoA desaturase-1; SREBP-1c, sterol regulatory element binding protein-1c; PPARγ, peroxisome proliferator-activated receptor γ; DGAT1, diglyceride acyltransferase; UCP, mitochondrial uncoupling proteins; ACOX1, peroxisomal acyl-coenzyme A oxidase 1; PPAR<sup>α</sup>, peroxisome proliferator-activated receptor α; ACS1, acetyl CoA synthetase 1; CPT1b, carnitine palmitoyltransferase 1b.
