PHYTOTHERAPY RESEARCH

Phytother. Res. (2017)

Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/ptr.5892

Anti-Obesity Activity of Saringosterol Isolated

from Sargassum muticum (Yendo) Fensholt
Extract in 3T3-L1 Cells

Jung A Lee, Young-Rak Cho, Seong Su Hong and Eun-Kyung Ahn*

Bio-Center, Gyeonggido Business and Science Accelerator, Gwanggyo-ro 147, Yeongtong-gu, Suwon-si, Gyeonggi-do 16229, Republic
of Korea

Saringosterol, a steroid isolated from Sargassum muticum, a brown edible alga widely distributed on the sea-
shores of southern and eastern Korea, has been shown to exhibit anti-obesity effect. In this study, we investigated
the anti-obesity activity of saringosterol through various experiments. The inhibitory effect of saringosterol on
adipogenesis was evaluated via Oil Red O staining in 3T3-L1 preadipocytes. After confirming that saringosterol
is not cytotoxic to these cells by using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, the
effect of saringosterol on the expression of various adipogenesis-related genes was analyzed via quantitative real-
time polymerase chain reaction and western blotting. We demonstrated that saringosterol dose dependently
inhibited adipocyte differentiation and expression of adipogenic marker genes such as adipocyte fatty acid-
binding protein, adiponectin, resistin, and fatty acid synthase in 3T3-L1 cells. In addition, saringosterol signifi-
cantly inhibited the mRNA and protein expression of peroxisome proliferator-activated receptor γ and CCAAT
enhancer-binding protein α in 3T3-L1 cells. Collectively, these findings indicate that saringosterol isolated from
S. muticum exhibits anti-obesity effect by inhibiting the expression of adipogenic transcription factors and
marker genes and that it may be developed as a drug to suppress adipogenesis. Copyright © 2017 John Wiley
& Sons, Ltd.
Keywords: Sargassum muticum; saringosterol; adipogenic marker genes; peroxisome proliferator-activated receptor γ; CCAAT en-
hancer-binding protein α; 3T3-L1 cells.

expressed during the differentiation of preadipocytes into

INTRODUCTION
Obesity is a common metabolic disease caused by a
combination of endocrine disorders, environmental fac-
tors, and genetic susceptibility. It is characterized by an
excessive accumulation of fat in the body due to imbal-
ance between energy intake and consumption and is
usually associated with conditions such as diabetes,
hypertension, hyperlipidemia, and cardiovascular dis-
eases (Kopelman, 2000; Spiegelman and Flier, 2001;
Vaneckova et al., 2014). The impact of obesity on global
public health has grown in recent years.
Adipocytes play an important role in energy
homeostasis and lipid metabolism. The differentiation of
preadipocytes into adipocytes is regulated by various hor-
mones and transcription factors, as well as by the intracellu-
lar accumulation of fat (Spiegelman and Flier, 2001; Rosen
and Spiegelman, 2006). Peroxisome proliferator-activated
receptor γ (PPARγ) and CCAAT enhancer-binding
protein α (C/EBPα) are crucial transcription factors in
adipogenesis; PPARγ is a member of the PPAR subfamily
of nuclear non-steroid hormone receptors, while C/EBPα
is a leucine zipper transcription factor. They are highly
* Correspondence to: Eun-Kyung Ahn, Bio-Center, Gyeonggido Business
and Science Accelerator, Gwanggyo-ro 147, Yeongtong-gu, Suwon-si,
Gyeonggi-do, 16229, Republic of Korea
E-mail: aek@gbsa.or.kr
adipocytes (Tontonoz et al., 1994; Brun et al., 1996;
Morrison and Farmer, 2000) and have been suggested as
regulators of the expression of various proteins present in
adipocytes, including adipocyte fatty acid-binding protein
(aP2), adiponectin, resistin, and fatty acid synthase (FAS)
(Lowell, 1999; Rosen et al., 2000; Jeon et al., 2004).
Sargassum muticum is a brown edible alga that is
widely distributed on the seashores of southern and
eastern Korea. Previous studies have demonstrated that
S. muticum has anti-oxidant, anti-bacterial, and anti-
inflammatory properties (Kim et al., 2007; Yoon et al.,
2010; Chae et al., 2013). Saringosterol, a steroid isolated
from Sargassum sp., has been shown to inhibit the growth
of Mycobacterium tuberculosis (Wachter et al., 2001), as
well as exhibit anti-obesity effect through its lipase
inhibitory activity (Kim et al., 2014). Furthermore,
24(S)-saringosterol has been reported as a cholesterol-
lowering agent based on its liver X receptor-β agonist func-
tion (Chen et al., 2014). However, there is no report to date
on the anti-obesity effect of saringosterol involving PPARγ
and C/EBPα expression with regard to adipogenesis.
In this study, we investigated whether saringosterol
isolated from S. muticum exhibits inhibitory activity on
adipocyte differentiation and adipogenic mediator ex-
pression in preadipocytes. Our results demonstrated
that saringosterol reduced lipid accumulation and
inhibited 3T3-L1 preadipocyte differentiation by sup-
pressing the expression of PPARγ and C/EBPα.

Received 17 March 2017

Revised 27 June 2017
Copyright © 2017 John Wiley & Sons, Ltd. Accepted 21 July 2017
 J.A. LEE ET AL.
MATERIALS AND METHODS
Preparation of S. muticum extract. Whole plants of S.
muticum were provided by the Jeju Biodiversity Research
Institute, Jeju, Republic of Korea, in September 2013.
The identity of the plant material was established by one
of the authors (Seong Su Hong), and a voucher specimen
(G40) was deposited at the Bio-Center, Gyeonggido
Business and Science Accelerator, Suwon-si, Republic
of Korea. Dried whole plants (2 kg) were pulverized and
extracted with EtOH (3 × 15 L) at room temperature
(20–25°C) for 24 h.
Isolation and identification of saringosterol. The ethanol
extract of S. muticum was filtered, concentrated in vacuo,
suitably diluted with water, and partitioned with
n-hexane, CH2Cl2, EtOAc, and n-BuOH. The CH2Cl2
extract (14 g) was subjected to column chromatography
over silica gel (n-hexane/CHCl3, 1:1 to 0:1; CHCl3/ace-
tone, 70:1 to 2:1) to yield 11 fractions (G40-21-1–11).
The G40-21-9 (499 mg) fraction was separated on a C18
RP silica gel column (MeOH/H2O, 3:7 to 1:0) to afford
11 subfractions (G40-25-1–11). Subfraction G40-25-1
(208 mg) was further purified via preparative HPLC
(Shimadzu, Kyoto, Japan) by eluting with MeOH/H2O
(78:22) at 12 mL/min to yield saringosterol (23.1 mg).
Its NMR spectra were recorded on a Bruker Ascend III
700 spectrometer (Bruker, Rheinstetten, Germany) using
tetramethylsilane as an internal standard, with chemical
shifts expressed in δ. Electrospray ionization mass
spectra were obtained with a LTQ Orbitrap XL mass
spectrometer (Thermo Scientific, Waltham, MA, USA).
Saringosterol. White amorphous powder; 1H-NMR
(CDCl3, 700 MHz) δ 5.83 (1H, ddd, J = 17.5, 11.2,
4.2 Hz, H-28), 5.37 (1H, brs, H-6), 5.21 (1H, dd,
J = 17.5, 4.2 Hz, H-29), 5.16 (1H, dd, J = 11.2, 4.2 Hz, H-
29), 3.55 (1H, m, H-3), 1.03 (3H, s, Me-19), 0.95 (3H, d,
J = 6.3 Hz, Me-21), 0.92 (3H, d, J = 6.3 Hz, Me-26), 0.89
(3H, d, J = 6.3 Hz, Me-27), 0.69 (3H, s, Me-18); 13C–
NMR (CDCl3, 175 MHz) δ 37.2 (C-1), 31.7 (C-2), 71.8
(C-3), 42.3 (C-4), 140.8 (C-5), 121.7 (C-6), 31.9 (C-7),
31.9 (C-8), 50.1 (C-9), 36.5 (C-10), 21.1 (C-11), 39.7 (C-
12), 42.3 (C-13), 56.7 (C-14), 24.3 (C-15), 28.2 (C-16),
55.8 (C-17), 11.9 (C-18), 19.4 (C-19), 35.9 (C-20), 18.8
(C-21), 29.1 (C-22), 34.8 (C-23), 77.7 (C-24), 36.1 (C-
25), 16.5 (C-26), 17.6 (C-27), 142.6 (C-28), 112.9 (C-29);
electrospray ionization MS (positive mode) m/z 451
[M + Na]+. The structure of saringosterol is presented
in Fig. 1A (Sheu and Sung, 1991).
Apparatus and chromatographic conditions. HPLC
analysis was performed on a Waters Alliance (Waters
Co., Milford, MA, USA) system composed of a 2998
photodiode array detector and e2695 separation mod-
ule. The separation was achieved using a YMC-Triart
C18 column (250 × 4.6 mm ID, 5 μm particle size)
(YMC Co., Ltd., Kyoto, Japan). The mobile phase
consisted of water–trifluoroacetic acid (99.95:0.05; v/v)
(solvent A) and acetonitrile (solvent B). The elution
was performed using the following gradient: initial
Copyright © 2017 John Wiley & Sons, Ltd.
10:90 (A:B v/v); 20 min 0:100 (A:B v/v); 30 min 0:100
(A:B v/v). The mobile phase was prepared daily, filtered
through a 0.45 mm, WTP 0.5 mm membrane (Whatman,
Maidstone, UK), sonicated before use, and delivered at
a flow rate of 1.0 mL/min. The injection volume was
20 μL, and the column temperature was at 30°C. All
the operations, the acquiring and analysis of data, were
controlled by Empower™ 3 software (Waters Co., Mil-
ford, MA, USA). The HPLC chromatogram data in-
cluding saringosterol is presented in Fig. 1B. And
HPLC chromatogram data and 13C-NMR spectrum data
of saringosterol are shown Fig. 1C and D, respectively.
Cell culture and adipocyte differentiation. Mouse 3T3-
L1 cells were purchased from American Type Culture
Collection (ATCC, Manassas, VA, USA). The cells
were maintained in Dulbecco’s modified Eagle’s me-
dium (DMEM; ATCC) containing 10% bovine calf se-
rum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin
(Invitrogen, Carlsbad, CA, USA), in a humidified atmo-
sphere of 5% CO2 at 37°C. Two days after the cells
reached confluence, they were incubated with MDI
(1 μΜ dexamethasone (Calbiochem, Billerica, MA,
USA), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX;
Calbiochem), and 1 μg/mL insulin (Sigma-Aldrich, St.
Louis, MO, USA)) in DMEM containing 10% fetal bo-
vine serum (FBS; ATCC). On day 2, the medium was
changed to DMEM containing 1 μg/mL insulin and
10% FBS. The progression medium was then changed
to DMEM containing 10% FBS every 2 days (days 4
and 6). The cells were treated with different concentra-
tions of saringosterol from days 0 to 8.
Oil Red O staining. Eight days after induction of adipo-
cyte differentiation, 3T3-L1 cells were washed with
phosphate-buffered saline (PBS), fixed with 3.7% form-
aldehyde in PBS for 30 min, and washed twice with PBS
and 70% ethanol. The cells were then stained with Oil
Red O (Sigma-Aldrich) in isopropyl alcohol/distilled
water for 30 min and washed with 70% ethanol and
PBS. Stained lipid droplets were observed with a micro-
scope (TE2000-U; Nikon, Tokyo, Japan) and dissolved
in isopropyl alcohol containing 4% Nonidet P-40
(Sigma-Aldrich) before quantification with a microplate
reader (SpectraMax 190PC; Molecular Devices, Sunny-
vale, CA, USA) at 510 nm.
Measurement of triglyceride contents and glycerol re-
lease. 3T3-L1 adipocytes were incubated in the presence
of saringosterol at concentrations of 50, 100, and 200 μM
in 24-well plates during 8 days after the initiation of dif-
ferentiation. Triglyceride (TG) contents were deter-
mined using triglyceride assay kit according to the
manufacturer’s instructions (Zen-Bio, Research Triangle
Park, NC, USA) with a microplate reader at 540 nm. And
glycerol release levels were measured using the free glyc-
erol assay kit according to the manufacturer’s protocol
(BioVision, Milpitas, CA, USA). The differentiated
3T3-L1 cells treated in DMEM with saringosterol of 50,
100, and 200 μM for 24 h. The media were collected
and assayed for glycerol with a microplate reader at
570 nm.
Phytother. Res. (2017)
 ANTI-OBESITY ACTIVITY OF SARINGOSTEROL

Figure 1. HPLC chromatogram of saringosterol. (A) Structure of saringosterol isolated from S. muticum. (B) HPLC chromatogram (205 nm)
13
of S. muticum extract. (C) HPLC chromatogram (205 nm) of saringosterol. (D) C-NMR spectrum of saringosterol (CDCl3, 175 MHz).
[Colour figure can be viewed at wileyonlinelibrary.com]

Cell viability assay. 3T3-L1 cell viability was determined
using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazo-
lium bromide (MTT; Duchefa Biochemie, Haarlem,
The Netherlands) assay. Cells were plated on 96-well
plates at a density of 5 × 103 cells per well in culture me-
dium. After 24 h, a time zero control plate was made.
Copyright © 2017 John Wiley & Sons, Ltd.
Saringosterol (12.5–200 μM) was applied directly, and
the cells were incubated for 24 h in a humidified 5%
CO2 atmosphere at 37°C. The MTT solution (5 mg/mL
in PBS) was then added to each well, followed by incu-
bation for 3 h. The medium was removed from the wells
by aspiration. Subsequently, 0.1 mL of buffered
Phytother. Res. (2017)
 J.A. LEE ET AL.

dimethyl sulfoxide was added to each well, and the plate

was shaken to dissolve the formazan. Absorbance was
measured with a microtiter plate reader at 540 nm.
RNA isolation and quantitative real-time PCR analysis.
Differentiated 3T3-L1 cells were harvested and washed
with PBS. Total RNA was extracted using Trizol reagent
(Invitrogen) and quantified with a spectrophotometer
(SD2000; Bioprince™ Technology Development Co.,
Seoul, Korea) by measuring the absorbance at 260 and
280 nm. cDNA synthesis was performed using 1 μg of
total RNA with the SuperScript® III first-strand synthe-
sis system (Invitrogen). The synthesized cDNA was am-
plified using specific primers with the SYBR Premix Ex
Taq system (TaKaRa, Shiga, Japan). The primers used
in the experiments are shown in Table 1. Glyceralde-
hyde 3-phosphate dehydrogenase was used as the in-
variant control.
Western blot analysis. 3T3-L1 cells were lysed on ice for
30 min in RIPA buffer containing a cocktail of protease
inhibitors (Sigma-Aldrich). The cells were centrifuged
at 16,000g for 30 min at 4°C to obtain the supernatant.
The cell lysates (50 μg) were separated on an 8% SDS
polyacrylamide gel and transferred onto nitrocellulose
membranes (Whatman, St. Louis, MO, USA). Protein
expression was analyzed by immunoblotting with anti-
bodies against PPARγ, C/EBPα (1:1000; Cell Signaling
Technology, Danvers, MA, USA), and β-actin (1:1000;
Cell Signaling Technology). Horseradish peroxidase-
conjugated anti-rabbit antibodies (1:5000; Cell Signaling
Technology) were used as secondary antibodies. The
proteins were detected with SuperSignal® West Pico
chemiluminescent substrate (Thermo Scientific) using
the Amersharm Imager 600 (GE Healthcare Life Sci-
ences, Chicago, IL, USA).
RESULTS
Effects of saringosterol on cell viability and lipid
accumulation in 3T3-L1 cells
We first examined the cytotoxicity of saringosterol in
3T3-L1 cells. The cells were treated with increasing
doses (12.5–200 μM) of saringosterol, and cell prolifera-
tion was determined using MTT assay. The different
concentrations of saringosterol were not cytotoxic to
3T3-L1 cells (Fig. 2A). Subsequently, the inhibitory ef-
fect of saringosterol on adipocyte differentiation was
assessed using MDI differentiation medium (containing
dexamethasone, IBMX, and insulin) to induce the dif-
ferentiation of 3T3-L1 preadipocytes. After 8 days, cells
treated with 50, 100, and 200 μM saringosterol were
stained with Oil Red O and observed under a micro-
scope. Saringosterol treatment significantly suppressed
adipocyte differentiation in a dose-dependent manner.
Lipid droplet accumulation in 3T3-L1 adipocytes
treated with 50, 100, and 200 μM saringosterol de-
creased to 56.5, 47.3, and 26.6%, respectively, relative
to that in untreated cells (Fig. 2B and C). Also, TG con-
tents were observed in 3T3-L1 cells (Fig. 2D). TG accu-
mulation was reduced with 50, 100, and 200 μM of
saringosterol compared with differentiated adipocytes.
We determined the effect of saringosterol on glycerol
release in the media of 3T3-L1 cells. As shown in
Fig. 2E, saringosterol increased glycerol release levels
in 3T3-L1 cells. These results clearly indicate that
saringosterol isolated from S. muticum inhibited lipid
and TG accumulation and induced glycerol secretion
in 3T3-L1 cells in a dose-dependent manner, without in-
ducing cytotoxicity.
Effect of saringosterol on the expression of PPARγ and
C/EBPα at mRNA and protein levels in 3T3-L1 cells

Both PPARγ and C/EBPα play important roles in cellu-

Statistical analysis. Data are expressed as means ± stan-
dard deviation (SD). Experimental results were ana-
lyzed for statistical significance using Student’s t-test
and one-way analysis of variance. Values of *p < 0.05
and **p < 0.01 were considered statistically significant.
lar responses related to lipid accumulation and adipo-
cyte differentiation (Gregoire et al., 1998; Rosen et al.,
2002). In view of the inhibitory activity of saringosterol
on adipocyte differentiation, we examined the expres-
sion of PPARγ and C/EBPα in 3T3-L1 cells using

Table 1. Primer sequences used for real-time PCR

Target Primer sequences Accession no.

GAPDH 50 -GTATGACTCCACTCACGGCAAA-30 (sense) BC083080

50 -GGTGTGGCTCCTGGAAGATG-30 (antisense)
PPARγ 50 -CGCTGATGCACTGCCTATGA-30 (sense) NM_011146
50 -AGAGGTCCACAGAGCTGATTCC-30 (antisense)
C/EBPα 50 -AGGTGCTGGAGTTGACCAGT-30 (sense) BC058161
50 -CAGCCTAGAGATCCAGCGAC-30 (antisense)
aP2 50 -CATGGCCAAGCCCAACAT-30 (sense) NM_024406
50 -CGCCCAGTTTGAAGGAAATC-30 (antisense)
Adiponectin 50 -AGCCTGGAGAAGCCGCTTAT-30 (sense) NM_009605
50 -TTGCAGTAGAACTTGCCAGTGC-30 (antisense)
Resistin 50 -TCAACTCCCTGTTTCCAAATGC-30 (sense) NM_022984
50 -TCTTCACGAATGTCCCACGA-30 (antisense)
FAS 50 -CTGAGATCCCAGCACTTCTTGA-30 (sense) NM_007988
50 -GCCTCCGAAGCCAAATGAG-30 (antisense)

Copyright © 2017 John Wiley & Sons, Ltd. Phytother. Res. (2017)
 ANTI-OBESITY ACTIVITY OF SARINGOSTEROL

Figure 2. Effects of saringosterol on viability and adipocyte differentiation in 3T3-L1 cells. (A) 3T3-L1 cells were treated with increasing con-
centrations of saringosterol (12.5–200 μM) for 24 h. Cell viability was determined using MTT assay. (B and C) Eight days after the induction
of differentiation, the cells were stained with Oil Red O. (D) Quantification of triglyceride accumulation. (E) Glycerol release levels were mea-
sured using a free glycerol assay kit. Data are expressed as means ± SD of three replicates; *p < 0.05, **p < 0.01, compared with MDI-
treated cells. [Colour figure can be viewed at wileyonlinelibrary.com]

quantitative real-time PCR and western blot. As
shown Fig. 3, saringosterol treatment significantly
inhibited the expression of PPARγ and C/EBPα at
the mRNA level in 3T3-L1 cells. Likewise, we con-
firmed that saringosterol also suppressed the expres-
sion of PPARγ and C/EBPα at the protein level in
these cells (Fig. 4A and B). Furthermore, the inhibi-
tory effect of saringosterol on the protein expression
of PPARγ and C/EBPα was very similar in pattern
to that of its inhibition of the mRNA expression of
PPARγ and C/EBPα (Figs. 3 and 4).
2004). Therefore, we investigated the effect of
saringosterol on the expression of adipogenic marker
genes encoding the proteins aP2, adiponectin, resistin,
and FAS. On day 8, fully differentiated adipocytes were
treated with 100 and 200 μM saringosterol, and their
mRNA expression was analyzed using quantitative
real-time PCR. As shown in Fig. 5, saringosterol treat-
ment effectively inhibited mRNA expression of aP2,
adiponectin, resistin, and FAS. These findings suggest
that saringosterol prevents adipocyte differentiation
and lipid accumulation through suppression of
adipogenic-related marker genes.

Effect of saringosterol on the expression of adipogenic

marker genes in 3T3-L1 cells
DISCUSSION
During 3T3-L1 adipocyte differentiation, PPARγ and
C/EBPα regulate the expression of various genes in- In our preliminary study, we examined the anti-
volved in adipogenesis (Rosen et al., 2000; Jeon et al., obesity effect of the ethanol extract of S. muticum
Copyright © 2017 John Wiley & Sons, Ltd. Phytother. Res. (2017)
 J.A. LEE ET AL.
Figure 2. (continued)
and showed that it dose dependently inhibited lipid
accumulation in 3T3-L1 cells based on Oil Red O
staining result (data not shown). Isolation of the ac-
tive compound from this extract led to the identifica-
tion of saringosterol. Saringosterol has also previously
been isolated from other seaweeds such as S. fusi-
form, Lessonia nigrescens, and S. thunbergii (Wachter
et al., 2001; Kim et al., 2014; Xu et al., 2001).
Until recently, previous investigation about
saringosterol has mainly reported on its lipase inhibi-
tory activity with regard to its anti-obesity effect
(Kim et al., 2014). In the present study, we assessed
the anti-adipogenic effect of saringosterol in
Figure 3. Effect of saringosterol on the mRNA expression of peroxisome proliferator-activated receptor γ and CCAAT enhancer-binding pro-
tein α in 3T3-L1 cells. Post-confluent 3T3-L1 cells were differentiated in the absence or presence of saringosterol for 8 days. Gene expres-
sion of peroxisome proliferator-activated receptor γ and CCAAT enhancer-binding protein α was evaluated via quantitative real-time PCR.
Data are expressed as means ± SD of three replicates; **p < 0.01, compared with MDI-treated cells.
Copyright © 2017 John Wiley & Sons, Ltd.
preadipocytes. First, we showed that saringosterol re-
duced lipid and TG contents in 3T3-L1 cells during
adipocyte differentiation without inducing cytotoxicity.
And we observed that saringosterol increased the
glycerol release in 3T3-L1 adipocyte (Fig. 2). This
finding led us to investigate its effect on the expres-
sion of transcription factors associated with adipogen-
esis. The transcription factors PPARγ and C/EBPα
are crucial in the process of cell differentiation by
which preadipocytes become adipocytes; PPARγ stim-
ulates lipoprotein lipase expression and promotes
fatty acid uptake, while C/EBPα induces terminal ad-
ipocyte differentiation. These two factors remain ele-
vated during the rest of the differentiation process
and life of the mature adipocyte (Brun et al., 1996;
Morrison and Farmer, 2000; Rosen, 2005). Accord-
ingly, many reports have associated suppression of
adipocyte differentiation with the downregulation of
PPARγ and/or C/EBPα (Tontonoz et al., 1995; Kang
et al., 2003; Farmer, 2005; Ahn et al., 2012).
In the present study, we demonstrated the inhibi-
tory effect of saringosterol on the expression of
PPARγ and C/EBPα at mRNA (Fig. 3) and protein
(Fig. 4) levels in fully differentiated 3T3-L1 cells
and showed that the inhibition of mRNA and protein
expression of PPARγ and C/EBPα followed a similar
pattern (Figs. 3 and 4). In addition, saringosterol
treatment decreased the mRNA expression of
adipocyte-specific genes such as aP2, adiponectin,
resistin, and FAS (Fig. 5). The induction of aP2 is
linked with PPARγ activation in fat cells, and to-
gether with adiponectin, they are both detected in
the adipose tissue during adipocyte differentiation
(Tontonoz et al., 1994; Glatz et al., 1995). Resistin is
a polypeptide secreted by adipocytes that is involved
in the regulation of glucose homeostasis, while FAS
is a crucial enzyme in fatty acid synthesis (Steppan
et al., 2001; Rajala et al., 2003; Sul and Wang,
1998). Thus, inhibition of the expression of these
molecules is associated with anti-obesity activity.
In conclusion, this is the first report to demonstrate
that saringosterol isolated from S. muticum inhibited
adipocyte differentiation via downregulation of PPARγ,
C/EBPα, and adipogenic marker genes such as aP2,
adiponectin, resistin, and FAS. These findings suggest
that saringosterol may be developed as a drug candi-
date for the prevention of obesity-related diseases.
Phytother. Res. (2017)
 ANTI-OBESITY ACTIVITY OF SARINGOSTEROL

Figure 4. Effect of saringosterol on the protein expression of (A) peroxisome proliferator-activated receptor γ (PPARγ) and (B) CCAAT en-
hancer-binding protein α (C/EBPα) in 3T3-L1 cells. Post-confluent 3T3-L1 cells were differentiated in the absence or presence of saringosterol
for 8 days. Protein expression of PPARγ and C/EBPα was analyzed by western blot with antibodies against PPARγ and C/EBPα. Data are
expressed as means ± SD of three replicates; **p < 0.01, compared with MDI-treated cells.

Figure 5. Effect of saringosterol on adipocyte fatty acid-binding protein, adiponectin, resistin, and fatty acid synthase gene expression in
3T3-L1 cells. Post-confluent 3T3-L1 cells were differentiated in the absence or presence of saringosterol for 8 days. mRNA expression of
adipocyte fatty acid-binding protein, adiponectin, resistin, and fatty acid synthase was evaluated by quantitative real-time PCR analysis.
Data are expressed as means ± SD of three replicates; **p < 0.01, compared with MDI-treated cells.

Copyright © 2017 John Wiley & Sons, Ltd. Phytother. Res. (2017)
 J.A. LEE ET AL.
Acknowledgement
This research was supported by the High Value-Added Food Technol-
ogy Development Program (311027-03-3-CG000) of the Ministry for
Agriculture, Food and Rural Affairs, Korea.
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Conflict of Interest
The authors have no conflicts of interest to declare.
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