Anti‐obesity effect of Melandrium firmum Rohrbach extract in 3T3‐L1 cells and high‐fat diet‐induced obese C57BL/6N mice

Abstract In this study, we first investigated the influence of Melandrium firmum Rohrbach (MF) on the accumulation of lipid content in 3T3‐L1 cells and in vitro results showed that MF extraction suppressed the differentiation of 3T3‐L1 pre‐adipocytes in a concentration‐dependent manner without showing cytotoxicity. Hence, we studied the effects of MF on preventing obesity in C57BL/6N mice. The results showed that MF decreased food efficiency ratio, body weight, epididymal adipose and hepatic tissue weight, hepatic lipid metabolites, and triacylglycerol and cholesterol serum levels, when compared with the high‐fat diet group. Moreover, MF significantly inhibited the expression of genes related to adipogenesis, such as PPAR‐γ, C/EBP‐α, and aP2, and those related to lipogenesis, such as SREBP‐1c, FAS, SCD‐1, and CD36 in epididymal adipose and liver tissues. These anti‐adipogenic and anti‐lipogenic effects of MF suggest that it could be used as a food including potential functional ingredient to prevent high‐fat diet‐induced obesity.


| INTRODUC TI ON
In the industrialized world, obesity is one of the epidemics associated with an increased risk of other life-threatening pathologies (Ravinet Trillou et al., 2003). Obesity results from the disequilibrium in energy intake and expenditure, and is known to be a risk factor for type 2 diabetes (Ono, Hattori, Fukaya, Imai, & Ohizumi, 2006). Obesity is caused by the generation of new adipocytes (You, Lee, Kim, Kim, & Chang, 2014), and adipogenesis is a process of cell differentiation where pre-adipocytes mature into adipocytes (Wang, Hwang, Kim, & Lim, 2017). Adipogenesis is a complex process that is accompanied by changes in morphology, hormones, and gene expression. CCAAT/enhancer-binding protein (C/EBP) transcription factor family and peroxidase proliferator-activated receptor γ (PPAR γ) act to regulate adipocyte differentiation (Lee et al., 2010), sterol regulatory element-binding protein  acts as a stimulator (Ali, Hochfeld, Myburgh, & Pepper, 2013), and adipocyte P2(aP2) and fatty acid synthase (FAS) also play a role in the maturation of adipocytes (Choi et al., 2000). Lipogenesis involves fatty acid and triglyceride synthesis in the liver, and fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and fatty acid esterification transcriptional factors like stearoyl-CoA desaturase (SCD-1) play an important role in lipogenesis in the adipose and liver tissues (Wang et al., 2017).

Melandrium firmum Rohrbach (MF) is a widely distributed plant in
Korea (Zheng et al., 2008). The dried aerial part of MF has been used as a medicine for acute nephritis and liver cirrhosis in China, and to treat breast cancer and lactation disorders in Korea (Zhang et al., 2015). MF contains several classes of compounds like sapogenin, saponin, flavonoids, and triterpenoids, and their pharmacological effects have been evaluated (Lee et al., 2012). The root extract of MF showed apoptotic effects in SHSY5Y neuroblastoma cells, and butanol fraction of the methanol extraction of whole body of MF exhibited anti-inflammatory activity. The methanol extract of MF inhibited the development of benign prostatic hyperplasia (BPH) in rats (Chandra & Rawat, 2015).
In this study, we examined the anti-adipogenic effects of MF extract using Oil Red O staining in 3T3-L1 adipocytes. In addition, the anti-obesity effects of the extract were investigated by measuring body weight gain, serum and lipid profiles, histological variation in the adipose tissue, and expression of the genes associated with adipogenesis and lipogenesis for 10 weeks in high-fat diet-fed obese mice.

| Plant material and preparation of the extract
The whole plant of MF was purchased from Jeongjin Distribution Co, Korea. The dried MF (1.5 kg) was pulverized, and the extract was obtained using 70% ethanol (15 L) at 25℃ for 48 hr. The MF extracts were filtered using filter paper (Hyundai Micro No. 20) and concentrated by a reduced pressure evaporator (N-1000;Tokyo Rikakikai) to obtain the extract powder.

| Oil Red O staining and determination of lipid content
Cells were stained with Oil Red O solution (Sigma-Aldrich), to investigate both adipogenic potential and lipid accumulation. On day 8, the cultured 3T3-L1 cells were washed with cold phosphate-buffered saline (PBS) and then fixed with 10% formaldehyde at 25℃. The cells were stained with filtered 0.5 μg/ml Oil Red O solution (0.5 g of Oil Red O in 500 ml of isopropyl alcohol) and washed twice. The lipid droplets were dissolved in isopropanol, and absorbance was measured at 540 nm using a microplate reader (Sensident scan, Labsystems).

| Animals and their diet
Mice followed the experimental diet for 10 weeks. Body weight was measured twice per week, and food intake was recorded daily.

| Collection of serum and tissue samples
After 10 weeks, all mice were sacrificed following a 12-hr fasting, and tissues were collected for analysis. Blood was collected from the inferior vena cava and was subjected to centrifugation at 2,090 g at 4°C for 15 min to separate the serum. The epididymal adipose tissue and liver were removed, weighed, and stored at 80°C until analysis.

| Histological analysis
Epididymal adipose tissue was fixed with 4% formaldehyde and embedded in paraffin. Sections (5 μm thick) were cut, and each section was stained with hematoxylin and eosin (H & E). All the sections were photographed using an optical microscope (Leica RM2235, Wetzlar, Germany) and printed at a final magnification of 200×. Images were observed using a microscope (Axiomager), and the diameter of each adipocyte was analyzed using the AxioVisionRel. 4.8 software (Zeiss).

| RNA extraction, cDNA synthesis, and realtime PCR
Total RNA was extracted from the epididymal adipose tissue using an Easy-Blue kit (Intron Biotechnology Inc) according to the protocol provided by the manufacturer. Then, total RNA was quantified with a NanoDrop-2000 (Thermo Fisher Scientific). cDNA was synthesized (an equal amount of total RNA) with the Moloney murine leukemia virus transcriptase and Oligo (dT) 15 primers (Promega) using a Life Touch thermal cycler (Life Eco, Bioer Technology). The program was set for 1 hr of initiation at 42°C, followed by 10 min of incubation at 95°C and 10 min at 4°C. RT-PCR was performed using the QuantiTect SYBR Green PCR kit (Qiagen), according to the manufacturer's instructions. cDNA was amplified for 40 cycles of denaturation (95°C for 30 s), annealing (57°C for 40 s), and extension (72°C for 40 s) using a RotorGene RG3000 real-time PCR machine (Corbett Research). The purity of the PCR product was determined using melting curve analysis. The relative quantification of the expression of each gene was calculated using the comparative threshold cycle (Ct) method (Applied Biosystems). mRNA levels were normalized to β-actin. Primer sequences are shown in

| Statistical analysis
Data from individual experiments are expressed as a mean value ± standard error (SE). Comparisons were carried out using a Student's unpaired t test and a one-way analysis of variance (ANOVA), as deemed appropriate. p < .05 was considered statistically significant.

| Effect of MF extract on cell viability of preadipocytes
To determine the cell viability, an MTS assay was performed by treating 3T3-L1 cells with MF extract (10 and 50 μg/ml). As shown in Figure 1a, MF showed no significant adverse effect on viability after 24 hr, indicating a noncytotoxic effect of MF on 3T3-L1 cells.

| Inhibitory effect of MF extract on lipid accumulation in 3T3-L1 cells
The anti-adipogenic effect of MF extract was evaluated using Oil

| Changes in body weight, food intake, and food efficiency ratio (FER)
In previous experiments, MF extract showed noncytotoxic and anti-adipogenic effects on 3T3-L1 cells. Hence, we decided to use MF extract for further in vivo anti-obesity studies. The compositions of experimental diets are listed in Table 1. Figure 2a shows the difference in body weight. There was no significant difference during the early weeks, but after 10 weeks on the experimental diet, the body weight of HFD mice increased by 2.09-fold than that of NFD mice. MF-supplemented group (at 10 weeks) showed a significant decrease in body weight, by 1.08-fold relative to the HFD group. Food intake showed no significant difference initially; however, after 3 weeks, there was a change in food intake in the MFsupplemented group (Figure 2b). As shown in Table 3, body weight gain was affected by MF supplementation and was associated with a significant decrease in food intake. However, the food efficiency ratio (FER) of the MF group showed only a 1.24-fold increase compared the HFD group during the 10-week feeding period. HDL cholesterol levels were lower in the MF group relative to the HFD group, but HTR (HDL cholesterol/ total cholesterol ratio) was not significantly different. The hepatic and renal toxicity of MF was confirmed by serum AST, ALT, BUN, and CREA levels. Increased levels of AST, ALT, BUN, and CREA were found in the HFD group, but were decreased in the MF group, relative to the HFD group.

| Changes in weight and morphology of adipose tissue
The adipocyte size and adipose tissue weight were measured to investigate whether the weight-reducing effect of MF was due to a decrease in fat mass. Significantly increased weight of the epididymal white adipose tissue was detected in the HFD group, when compared with the NFD group (Table 3), and the adipocyte size was larger in the HFD group than in the NFD group (Figure 3).

| Effect of MF on the expression of genes related to adipogenesis and lipogenesis in white adipose tissue
To evaluate MF-mediated reduction in adipocyte size, mRNA levels of genes related to adipogenesis and lipogenesis were measured. As shown in Figure 4, MF administration significantly down-regulated the expression of PPAR-γ and fatty acid-binding protein 4 (aP2), which are involved in adipogenesis. The expression of lipogenic genes such as FAS and SCD-1 was also reduced by MF administration, but the difference was not significant.

| Change in liver weight and hepatic lipid metabolites
We examined the effect of MF on the change in liver weight and hepatic lipid metabolites in HFD-fed mice because obesity may be F I G U R E 2 Effects of Melandrium firmum Rohrback extract on (a) body weight and (b) food intake in high-fat dietinduced obese mice. Results are presented as mean ± SE (n = 6). NFD, normal-fat diet control; HFD, high-fat diet control; GR, HFD + 1% Garcinia cambogia extract; MF, HFD + 1% Melandrium firmum Rohrback one of the causes of development of a fatty liver. The liver weight in the HFD group was higher than in the NFD group, and MF administration decreased it by 1.26-fold (Table 3). Hepatic lipid metabolites (fatty acids, phospholipid, lipid moieties, and cholesterol) were analyzed, and their levels were found to be increased in the HFD group (Table 5). MF administration decreased the levels of hepatic lipid metabolites, when compared with the HFD group. These results suggest that MF administration may inhibit lipid accumulation in the hepatic tissue.

| Effect of MF on the expression of genes related to lipogenesis in the liver
To examine the effect of MF administration on the decline of hepatic lipid accumulation, we measured the mRNA levels of genes related to lipogenesis. MF administration significantly decreased the expression of lipogenesis-related gene FAS, and the other genes that showed a decrease in the mRNA levels relative to the HFD group were SREBP-1c, SCD-1, and fatty acid translocase (CD36) in the hepatic tissue ( Figure 5).

| D ISCUSS I ON
Obesity is a costly-to-treat condition and may be associated with negative social impression (Kwon et al., 2003); it may be associated with other metabolic disorders including type 2 diabetes and cardiovascular diseases (Kang et al., 2013). The ideal anti-obesity drug is the once that leads to a sustained weight loss with minimal side effects (Rodgers et al., 2012). The cellular development of obesity-associated adipose tissue involves both hyperplasia and hypertrophy (Ross & Desai, 2013  in obesity (Wong, Kaneda, & Morita, 2014).
A number of reports have indicated that PPAR-γ, C/EBP-α, and aP2 are the major genes related to adipogenesis, while FAS and SCD-1 are related to lipogenesis. Among them, PPAR-γ is a central regulator of fat cell differentiation, and this gene is linked aP2 as adipose-specific enhancer. Moreover, PPAR-γ interacts with another transcription factor, C/EBP-α, that is induced in adipogenesis and itself shows an adipogenic action (Spiegelman, 1998).
FAS and SCD-1 are induced by these transcription factors (Wang, Hudak, & Sul, 2010), and they are the key enzymes involved in fatty acid metabolism responsible for synthesizing palmitate (C16:0) (Wang et al., 2017). We performed RT-PCR to analyze the change in adipogenesis and lipogenesis due to MF extract. In this experiment, MF reduced the expression of these genes PPAR-γ, C/EBP-α, aP2, FAS, and SCD-1. These results suggest that MF administration suppressed adipogenesis and lipogenesis by decreasing these transcription factors in the adipose tissue.
The liver fat content reflects lipogenesis (Adiels et al., 2006), and hepatic tissue weight is an important target for the   F I G U R E 5 Effects of Melandrium firmum Rohrback extract on the mRNA levels of lipogenesis-related genes in the liver. Results are presented as mean ± SE. An asterisk indicates a significant difference (**, p < .01). NFD, normal-fat diet; HFD, high-fat diet; GR, HFD + 1% Garcinia cambogia extract; MF, HFD + 1% Melandrium firmum Rohrback

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interest to declare.

E TH I C A L A PPROVA L
This study was approved by the Institutional Review Board of Hallym University.