**4. Discussion**

In recent years, many side e ffects have been reported for drugs used for therapeutic purposes, and numerous clinical studies have examined natural products and natural product-derived compounds [26,27]. In this study, we confirmed the anti-obesity e ffect of three natural materials. In previous studies based on these materials, *N. nucifera* L was found to be a medicinal plant that is not only useful for treating gastritis, bleeding, diarrhea, hemorrhoids, and enuresis, but also contains various biologically active components such as polyphenolics, flavonoids, and tannic acid [28]. SOD, CAT, GST played as defence means against the reactive oxygen species in biological systems. TBARS are formed as a byproduct of lipid peroxidation. This material was reported to have anti-oxidative activity by increasing the levels of SOD, CAT, GST and decreasing TBARS level in liver [29] and anti-obesity activity in HFD-induced mice or anti-obesity activity for inducing apoptosis in 3T3-L1 adipocytes, but few studies have examined these e ffects [30]. *Morus alba* L, which is prepared for medicinal use such as treating headache, fever, and cough, shows pharmacological activities, particularly antidiabetic effects on lowering blood sugar [31]. *Raphanus sativus* is an herbaceous plant belonging to the cruciferous family and has been reported to have excellent anti-obesity e fficacy [32]. Recent studies have demonstrated the anti-obesity activity of a single material, but the mechanism action of this material in a mixture has not been evaluated.

We selected EM01, which contains a high content of *Nelumbo nucifera* [16], among the 26 mixtures (13 kinds of hot water extracts and 13 ethanol extracts) through the antioxidant and anti-obesity experiments at in vitro model. EM01 treatment has an anti-obesity e ffect at in vivo model using C57BL/6J obese mice. The weight of HFD-induced mice was e ffectively lower and the weight of the adipose tissue was significantly decreased compared with the control.

Type 2 diabetes is one of the most common disabilities caused by obesity. Obesity directly a ffects insulin function, which causes glucose to enter the cell membrane for energy metabolism, resulting in insulin resistance and type 2 diabetes [33]. A glucose tolerance test was conducted to investigate the glucose processing ability. EM01 e ffectively lowered the blood glucose level. This result was similar to those of the glucose tolerance test using neferine an alkaloid compound extracted from *N. nucifera* seed [34].

EM01 reduced the serum lipid profile (TC, HDL-cholesterol, LDL-cholesterol, TG) of HFD-induced obese mice. It may be useful for preventing dyslipidemia induced by obesity [35]. AST, ALT, and creatinine are toxicity indicators of the liver and kidney [24]. EM01 reduced the levels of these markers in the serum and improved the function of each organ.

Adipocytes not only play a role as energy reservoirs but also regulate endocrine function. Adipokine, a hormone specifically expressed and secreted from adipocytes such as adiponectin and leptin, is involved in fatty acid and glucose metabolism. IGF-1 plays an important role in regulating adipose tissue growth. These adipokines interact closely with each other and regulate the hormonal system in the body [36]. EM01 significantly increased the adiponectin level and reduced leptin and IGF-1 level in serum. According to a previous study [37], glucose and lipid metabolism are regulated by insulin signaling. Glucose and fatty acid enter cells and are metabolized for glycogenesis and β-oxidation, respectively. Based on these results, our study confirms that EM01 improves the process of bringing glucose and fatty acid into the cells, resulting in anti-obesity and anti-diabetic e ffects.

Adipogenesis is the process of cell di fferentiation by which pre-adipocytes become adipocytes. PPARγ is a key factor in adipogenic transcription [1]. In this study, it was found that mRNA expression of PPARγ in the liver tissue was reduced by administration of EM01. Also, the expression levels of FAS, DGAT1, SCD-1, leptin, SREBP1c were decreased, while the expression levels of UCP2, PPAR α, ACOX1, and adiponectin increased in the EM01, Q3OG treatment group in the liver. These results sugges<sup>t</sup> that EM01 treatment inhibits adipogenesis of 3T3-L1 adipocytes and improves fatty acid oxidation. EM01 regulated lipid metabolism in epididymal adipose tissue and abdominal subcutaneous fat. Our results demonstrate that EM01 has an anti-obesity e ffect in 3T3-L1 adipocytes and C57BL/6J obese mice.
