Samnamul (Shoots of Aruncus dioicus ) Inhibit Adipogenesis by Downregulating Adipocyte-Speciﬁc Transcription Factors in 3T3-L1 Adipocytes

: Adipocyte-speciﬁc transcription factors and antioxidants are considered the best target of obesity. Aruncus dioicus var. kamtschaticus ( A. dioicus , Samnamul) is easily available owing to edible and inexpensive. However, the anti-adipogenic e ﬀ ects of the underlying mechanism of A. dioicus extract (ADE) have not yet been reported. In the present study, we evaluate anti-adipogenic pathway in 3T3-L1 adipocytes, antioxidant activities and quantiﬁed phenolics using high-performance liquid chromatography of ADE. The results revealed ADE had reduced adipocyte di ﬀ erentiation (0.72-fold vs. MDI (media of di ﬀ erentiation) control), triglyceride (TG; 0.50-fold vs. MDI control, p < 0.001), and total cholesterol contents (0.77-fold vs. MDI control) by regulating adipocyte-speciﬁc transcription factors (C / EBP α , PPAR γ , and SREBP1) and their downstream mRNA (AdipoQ, Ap2, SREBP1-c, and FAS) levels. Furthermore, ADE has higher total phenol and ﬂavonoid contents and scavenging assay in the DPPH and ABTS + . In particularly, ADE contains chlorogenic acid (7.04 mg / kg), ca ﬀ eic acid (20.14 mg / kg), ferulic acid (1.74 mg / kg), veratric acid (29.31 mg / kg), cinnamic acid (4.70 mg / kg), and quercetin (4.18 mg / kg). In conclusion, since these phenols, especially quercetin, in the ADE appear to reduce di ﬀ erentiation, TG and cholesterol content by regulating adipocyte-speciﬁc transcription factors in adipocytes, ADE has the potential to be developed into a new antioxidant and anti-obesity therapeutics.


Introduction
Obesity means an excessive accumulation of fat in the body by an increase in hypertrophy of adipose cells with no effects on the number of cells and causes chronic diseases including diabetes mellitus, cardiovascular disease, and some cancers [1,2]. In recent times, the prevalence rate of obesity has increased to the extent that it can be considered as a global syndemic such as epidemics and climate change [3]. These current trends in obesity carry a high personal cost and social costs for public policy [4]. Thus, anti-obesity studies are conducted to reduce the burden on various diseases caused by

Preparation of Sample
Aerial parts of A. dioicus (AD) were collected from the Department of Herbal Crop Research (Eumsung, Chungcheongbuk-do, Korea) in 2018. AD was freeze-dried after collection. For the preparation of extracts, AD (10 g) was ground, sifted through a testing sieve (aperture 1.40 mm, wire 0.71 mm). Dried AD was extracted with 70% ethanol (ADE) at a 1:10 (v:v) ratio for 24 h, three times at room temperature. After filtration, extracts were concentrated in vacuo used by vacuum evaporator (Rotavapor R-121, Buchi, Switzerland), freeze-dried (PVTFD50R, ilShinBioBase, Korea; 20 mTorr, −40 • C, 1 week), and stored at −80 • C. This study was approved by the Cooperative Research Program for Agriculture Science and Technology Development Program (PJ01361603), Rural Development Administration, Republic of Korea.

Cytotoxicity
Cell viability was measured with a CellTiter 96 Aqueous One Solution Cell Proliferation Assay Kit (Promega Corporation, Madison, WI, USA) according to the manufacture's instruction. Briefly, cells (1 × 10 4 cell/mL) were seeded in 96 well plates and incubated at 37 • C in 5% CO 2 with ADE   µg/mL). After 24 h, MTS solution was added to each well, incubated for 1 h, and absorbance at 490 nm was measured using a Synergy H1 microplate reader (Biotek, Winooski, VT, USA).

Oil Red O Staining
Lipid droplets were measured using Oil Red O (ORO; Sigma, St. Louis, MO, USA) stain as described by Ramirez-Zacarias et al. [16]. The ORO working solution was prepared with dH 2 O and ORO (6/4 = w/v). The 3T3-L1 cells were washed with phosphate-buffered saline (PBS) and were then fixed in 10% formaldehyde for 1 h incubated at 37 • C. Fixed cells were washed with ddH 2 O and then washed with 60% isopropanol for 3 min. The cells were stained with ORO solutions for 15 min. The cells were washed with dH 2 O four times to remove the un-stained dye. The stained droplet was observed with an inverted phase microscope (10×; Observer A1, Zeiss, Oberkochen, Germany). In addition, isopropanol was added for quantification to elute stained reagents, and the absorbance was measured at 520 nm using by Synergy H1 microplate reader.

Triglyceride (TG) Assay
3T3-L1 adipocytes in the 12 wells plates were rinsed with PBS. Cells were homogenized with 5% NP-40/ddH2O. TG in adipocytes was measured by a Triglyceride Assay Kit from Abcam (Cambridge, UK) according to the instruction of the manufacturer. The absorbance was evaluated at 570 nm by a Synergy H1 microplate reader.

Measurement of Total Cholesterol
Cells were lysed with chloroform/isopropanol/NP-40 (v/v = 7/11/0.1). Cells were centrifuged for 5 min at 15,000× g, and then the supernatant was transferred to a new tube. The supernatant was dried at 50 • C to remove chloroform. Dried supernatant was dissolved with assay buffer (included in Kit). Cellular cholesterol levels were determined with a Cholesterol Assay Kit from Abcam (Cambridge, UK) according to the instruction of the manufacturer. The absorbance was performed at 570 nm using a Synergy H1 microplate reader.

Western Blot
Whole adipocyte for protein extraction was lysed in a RIPA buffer containing protease and phosphatase inhibitor cocktail (GenDEPOT, Katy, TX, USA). Cell lysates containing equal amounts of proteins were determined using the Bradford assay (BioRad, Hercules, CA, USA). Western blot was performed as described previously reported [17]. The PVDF-membranes were incubated with primary antibody (1:500-1000 dilution) at 4 • C, overnight, and secondary antibody (1:2000 dilution) were treated for 2 h. All primary and secondary antibodies were prepared by cell signaling (Beverly, MA, USA). The blots were visualized using the enhanced chemiluminescence (ECL) reagent (BioRad, Hercules, CA USA). Quantitative analysis was measured with free ImageJ (version 1.52a for windows; NIH, Rockville, MD, USA).

Quantitative Real-Time Polymerase Chain Reaction (qPCR)
Total RNA of adipocytes was extracted by TRIzol reagent (Ambion, Austin, TX, USA) referring to the manufacturer's instructions. cDNA was synthesized using a reverse transcriptase premix kit (Elpis Biotech, Daejeon, Korea). The qPCR was performed using the QuantiTect SYBR Green PCR Kit (Qiagen, Valencia, CA, USA). Primers were designed using Primer-BLAST shown in Table 1 (NCBI, Bethesda, MD, USA). The threshold cycle (Ct) value for each gene was normalized by β-actin.

Measurement of Antioxidant Activity
DPPH (Sigma-Aldrich, MO, USA) scavenging assay was followed by Mishra et al. with some modifications [18]. The DPPH solutions were prepared by DPPH (300 µM) in 99.9% ethanol was prepared. The working solution was diluted with 99.9% ethanol. The ADE (40 µL) were allowed to react with the DPPH solution (160 µL) for 1 h in the dark. Then, the absorbance was taken at 515 nm and was expressed as the half-maximal inhibitory concentration (IC 50 , µg/mL).
The ABTS (Sigma-Aldrich, MO, USA) assay was slightly modified by Miller and Rice-Evans [19]. Briefly, the stock of ABTS solutions included ABTS + solution (7.4 mM) and potassium persulfate (2.6 mM) for 4 h at 4 • C. The ABTS + solution was diluted with dH2O until an absorbance of 0.7 ± 0.02 at 734 nm. Then, ADE (20 µL) was mixed with ABTS + solution (180 µL) for 1 h in the dark. The absorbance was performed at 734 nm and was expressed as the half-maximal inhibitory concentration (IC 50 , µg/mL).

Statistics
All experimental results are presented as the means ± standard deviation (SD). The statistical significance of differences in this study was determined by a one-way analysis of variance (ANOVA) using Tukey's multiple comparison test (Prism 5.02 GraphPad Software, San Diego, CA, USA).

ADE Has Anti-Adipogenesis Effect on 3T3-L1 Adipocyte
The viability of 3T3-L1 adipocytes did not change 25-100 µg/mL ADE (≥100%), while 200 µg/mL ADE (85.97%, p < 0.001) was cytotoxic ( Figure 1a). Therefore, ADE concentrations up to 100 µg/mL were used for subsequent experiments. The anti-adipogenic effect of ADE was determined using ORO during the differentiation. Microscopically, the MDI control group had more stained lipid droplets than the undifferentiated controls, and the stained lipid droplets of the ADE groups were decreased with 50 and 100 µg/mL (Figure 1b). The amount was also significantly increased in the differentiated controls compared to the undifferentiated controls (3.46-fold vs. control, p < 0.001) and the 50 and 100 µg/mL ADE groups had lower fat accumulation rates (0.90-and 0.72-fold vs. MDI control, p < 0.05 and p < 0.001). In particular, the level was lower in the 100 µg/mL ADE group than the positive controls (conjugated linoleic acids, CLA; 0.86-fold vs. MDI control, p < 0.001), shown in Figure 1c.

Statistics
All experimental results are presented as the means ± standard deviation (SD). The statistical significance of differences in this study was determined by a one-way analysis of variance (ANOVA) using Tukey's multiple comparison test (Prism 5.02 GraphPad Software, San Diego, CA, USA).

ADE Has Anti-Adipogenesis Effect on 3T3-L1 Adipocyte
The viability of 3T3-L1 adipocytes did not change 25-100 μg/mL ADE (≥100%), while 200 μg/mL ADE (85.97%, p < 0.001) was cytotoxic (Figure 1a). Therefore, ADE concentrations up to 100 μg/mL were used for subsequent experiments. The anti-adipogenic effect of ADE was determined using ORO during the differentiation. Microscopically, the MDI control group had more stained lipid droplets than the undifferentiated controls, and the stained lipid droplets of the ADE groups were decreased with 50 and 100 μg/mL (Figure 1b). The amount was also significantly increased in the differentiated controls compared to the undifferentiated controls (3.46-fold vs. control, p < 0.001) and the 50 and 100 μg/mL ADE groups had lower fat accumulation rates (0.90-and 0.72-fold vs. MDI control, p < 0.05 and p < 0.001). In particular, the level was lower in the 100 μg/mL ADE group than the positive controls (conjugated linoleic acids, CLA; 0.86-fold vs. MDI control, p < 0.001), shown in Figure 1c.

ADE Reduces the TG and Total Cholesterol Levels in 3T3-L1 Adipocytes
Exposure to MDI to cause the differentiation led to increases in the TG content compared to the control (16.91-fold vs. control, p < 0.001; Figure 2a). All ADE concentrations from 25 μg/mL inhibited

ADE Reduces the TG and Total Cholesterol Levels in 3T3-L1 Adipocytes
Exposure to MDI to cause the differentiation led to increases in the TG content compared to the control (16.91-fold vs. control, p < 0.001; Figure 2a). All ADE concentrations from 25 µg/mL inhibited TG accumulation. The TG levels were markedly lower in the 50 and 100 µg/mL ADE groups (0.79-and 0.50-fold vs. MDI control, p < 0.001). The level in the positive control (CLA, 50 µM) was 0.83-fold vs. MDI control (p < 0.01), which was similar to 50 µg/mL, but higher than 100 µg/mL ADE. The cholesterol level was significantly increased in the MDI control (2.05-fold vs. control, p < 0.001; Figure 2b). Treatment with 100 µg/mL ADE reduced cholesterol by 0.77-fold (p < 0.001) compared with MDI control and was lower than in the positive control (CLA, 50 µM; 1.03-fold vs. MDI control). Therefore, 50 and 100 µg/mL ADE were used to examine adipogenic-and lipogenic-related protein expression on 3T3-L1 adipocytes.

Antioxidant Activities of ADE
Oxidative stress promotes adipogenesis, which is the conversion of pre-adipocytes-to-adipocytes [27]. Thus, the antioxidant activity and phenol, flavonoid, and phenolic contents were measured to examine functional compounds that account for the antioxidant activity and anti-adipogenic and anti-lipogenic effects. The DPPH and ABTS+ scavenging assay, indicative of antioxidant activity, was assessed with a simple colorimetric method [23]. The

Antioxidant Activities of ADE
Oxidative stress promotes adipogenesis, which is the conversion of pre-adipocytes-toadipocytes [27]. Thus, the antioxidant activity and phenol, flavonoid, and phenolic contents were measured to examine functional compounds that account for the antioxidant activity and anti-adipogenic and anti-lipogenic effects. The DPPH and ABTS+ scavenging assay, indicative of antioxidant activity, was assessed with a simple colorimetric method [23]. The antioxidant activity of ADE was examined using ascorbic acid. The IC 50 values of ADE were 66.96 and 30.56 µg/mL for the ABTS+ and DPPH scavenging effects, respectively ( Table 2). The phenols and flavonoids have beneficial antioxidant effects [28], so TPC and TFC were measured. The ADE contained 127.39 mg/g TPC and 104.17 mg/g TFC. Phenolic compounds responsible for the beneficial effects of ADE were analyzed by HPLC ( Figure 5). To determine the phenolic compounds, ADE was re-extracted in phenol-rich fractions. The phenolic compounds are shown in Table 3. Only chlorogenic (7.04 ± 1.10 mg/kg), caffeic (20.14 ± 0.52 mg/kg), ferulic (1.74 ± 0.87 mg/kg), veratric (29.31 ± 4.26 mg/kg), and cinnamic (4.70 ± 0.86 mg/kg) acids and quercetin (418.41 ± 7.26 mg/kg) were detected in the ADE. Therefore, these compounds likely play a crucial role in the anti-adipogenic effects of ADE. Phenolic compounds responsible for the beneficial effects of ADE were analyzed by HPLC ( Figure 5). To determine the phenolic compounds, ADE was re-extracted in phenol-rich fractions. The phenolic compounds are shown in Table 3. Only chlorogenic (7.04 ± 1.10 mg/kg), caffeic (20.14 ± 0.52 mg/kg), ferulic (1.74 ± 0.87 mg/kg), veratric (29.31 ± 4.26 mg/kg), and cinnamic (4.70 ± 0.86 mg/kg) acids and quercetin (418.41 ± 7.26 mg/kg) were detected in the ADE. Therefore, these compounds likely play a crucial role in the anti-adipogenic effects of ADE.

Discussion
This study explored the anti-obesity potential of ADE by regulating the expression of adipogenic proteins and mRNA, such as C/EBPα, PPARγ, AdipoQ, and Ap2, and the lipogenic proteins and mRNA, including SREBP1c and FAS in 3T3-L1 adipocytes. The ADE extract had antioxidant activity, as shown with the ABTS and DPPH scavenging assays, and antioxidants were detected in the TPC, TFC, and HPLC analyses.
Obesity is associated with adipocyte differentiation and lipid accumulation [29]. Adipogenesis is the differentiation of pre-adipocytes into mature adipocytes, which have a large internal lipid droplet and store TG [30]. When ADE was treated in the process of differentiation of 3T3L1 adipocytes, lipid droplets and TG were effectively decreased (Figures 1 and 2). This result is seen as a result of reducing adipogenesis. To evaluate the protein expression of adipogenic-related factors, adipocyte-specific transcription factors such as C/EBPα, PPARγ, and SREBP-1 were identified [31]. The activation of adipogenesis involves the cooperative interplay of members of the C/EBP and PPAR families, especially C/EBPα and PPARγ. They promote adipocyte differentiation by activating the transcription of the other synergistically [6]. C/EBPα and PPARγ activate downstream genes like adipoq and ap2 at the terminal stage of differentiation to cause fat accumulation in cells [24]. SREBP-1 controls lipogenesis and lipid homeostasis [30] by mediating the induction of lipid synthesis, cholesterol synthesis, and lipogenic enzyme genes such as srebp-1c and fas [7]. ADE reduced the total cholesterol content by inhibiting the expressions of the SREBP-1, srebp-1c, and fas factors involved in cholesterol synthesis (Figure 4) in adipocytes.
One study reported a correlation between antioxidants and obesity prevalence and found that free radicals promote the conversion of pre-adipocytes into mature adipocytes [31]. DPPH and ABTS free radical scavenging is a simple, convenient method for assessing antioxidant activity in food and complex samples [32]. As shown in Table 1, the free radical scavenging capacity (IC 50 ) of ADE was 66.96 µg/mL in DPPH and 30.56 µg/mL in ABTS, which are better than the values of other medicinal plants [33]. Some phenols and flavonoids exert antioxidant activity and anti-obesity effects by lowering TG and cholesterol levels, reducing adipogenic transcription factors, and increasing fat oxidation [34]. As mentioned above, ADE contained 127.39 mg/g total phenols and 104.17 mg/g total flavonoids (Table 2). Using HPLC, ADE was found to contain the compounds shown in Figure 5. Of these, the quercetin levels were highest (418.41 mg/kg) in ADE (Table 3). Quercetin reduces lipid accumulation in obese mice by increasing heme oxygenase-1, an antioxidant enzyme, and downregulating the adipocyte-specific transcription factors C/EBPα and PPARγ [35,36]. Quercetin in ADE likely plays a critical role in downregulating adipocyte-specific transcription factors and generating antioxidant activity.

Conclusions
ADE prevents adipogenesis and lipogenesis processes in 3T3-L1 adipocytes by suppressing lipid droplets, TG, and cholesterol levels. ADE acts by regulating transcription factors, C/EBPα, PPARγ, SREBP-1, and their target mRNAs, such as SREBP-1c, FAS, AdipoQ, and Ap2. ADE affects ABTS and DPPH scavenging and contains phenols and flavonoids such as caffeic, ferulic, veratric, and cinnamic acids and quercetin, which have critical roles in its anti-adipogenic and -lipogenic effects. Therefore, ADE may be a useful functional food for treating and obesity and a source of new antioxidants and anti-obesity therapeutics.