Anti-adipogenic activity of compounds isolated from Idesia polycarpa on 3T3-L1 cells

Article abstract of DOI:10.1016/j.bmcl.2013.04.011

Recently, obesity is a complex multifactorial chronic disease increasing the risk for type 2 diabetes, coronary heart disease and hypertension, and has become a major worldwide health problem. In the course of screening natural products employing 3T3-L1 cells as an in vitro system, the methanol extract of Idesia polycarpa Maxim. Fruits (Flacourtiaceae) significantly inhibited adipocyte differentiation by measuring lipid contents using oil red O staining. One new compound, 6-(oxymethyl)-2-hydroxyphenyl-O-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranoside (8), was isolated along with nine known compounds (1-7 and 9-10) from CHCl₃ and n-BuOH fractions of the methanol extract of I. polycarpa fruits. Among them, idescarpin (1) with 1-hydroxy-6-oxo-2-cyclohexenecarboxylate moiety showed the most potent inhibitory activity on adipocyte differentiation with IC₅₀ values of 23.2 μM. Idescarpin (1) dramatically suppressed the induction of C/EBPα expression, whereas it significantly increased the induction of PPARγ expression, supported by quantitative real time PCR and Western blot analysis. The down-regulation in mRNA levels of SREBP1c, SCD-1, and FAS by idescarpin (1) during adipocyte differentiation revealed that the inhibition of adipocyte differentiation was mediated by the regulation of lipogenesis. Taken together, we suggest that idescarpin (1) shows a great potential against obesity and diabetes though the anti-adipogenic activity and the up-regulation of PPARγ.

Full text of DOI:10.1016/j.bmcl.2013.04.011

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3170–3174
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Anti-adipogenic activity of compounds isolated from Idesia
polycarpa on 3T3-L1 cells
a
a
b
b
c
a,
⇑
Mina Lee , Hyang Hwa Lee , Jin-Ku Lee , Sang-Kyu Ye , Seung Hyun Kim , Sang Hyun Sung
a
College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National University, Daehak-Dong, San 56-1, Sillim-Dong, Gwanak-Gu, Seoul 151-742, Republic of Korea
Department of Pharmacology, Seoul National University, College of Medicine, 28 Yongun-Dong, Chongro-Gu, Seoul 110-799, Republic of Korea
College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon 406-840, Republic of Korea
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Recently, obesity is a complex multifactorial chronic disease increasing the risk for type 2 diabetes, cor-
onary heart disease and hypertension, and has become a major worldwide health problem. In the course
of screening natural products employing 3T3-L1 cells as an in vitro system, the methanol extract of Idesia
polycarpa Maxim. Fruits (Flacourtiaceae) significantly inhibited adipocyte differentiation by measuring
Received 9 November 2012
Revised 25 March 2013
Accepted 3 April 2013
Available online 10 April 2013
lipid contents using oil red
O-b- -glucopyranosyl-(1 ? 6)-b-
1–7 and 9–10) from CHCl and n-BuOH fractions of the methanol extract of I. polycarpa fruits. Among
them, idescarpin (1) with 1-hydroxy-6-oxo-2-cyclohexenecarboxylate moiety showed the most potent
inhibitory activity on adipocyte differentiation with IC50 values of 23.2 M. Idescarpin (1) dramatically
suppressed the induction of C/EBP expression, whereas it significantly increased the induction of PPAR
O
staining. One new compound, 6-(oxymethyl)-2-hydroxyphenyl-
D
D-glucopyranoside (8), was isolated along with nine known compounds
Keywords:
(
3
Idesia polycarpa
Flacourtiaceae
Adipogenesis
Adipocyte differentiation
Adipokine
l
a
c
expression, supported by quantitative real time PCR and Western blot analysis. The down-regulation in
mRNA levels of SREBP1c, SCD-1, and FAS by idescarpin (1) during adipocyte differentiation revealed that
the inhibition of adipocyte differentiation was mediated by the regulation of lipogenesis. Taken together,
we suggest that idescarpin (1) shows a great potential against obesity and diabetes though the anti-adi-
Obesity
3
T3-L1 cells
pogenic activity and the up-regulation of PPARc.
Ó 2013 Elsevier Ltd. All rights reserved.
Obesity is a serious topic in public health related problems ta-
desaturase 1 (SCD-1) regulate the expression of genes involved in
8
,9
ken both independently and in association with various patholog-
ical disorders, such as hypertension, atherosclerosis, Type II
lipogenesis and fatty acid desaturation. The cellular and molecu-
lar mechanisms of adipocyte differentiation have been extensively
studied using preadipocyte culture systems. Committed preadipo-
cytes undergo alteration of cell shape, growth arrest, clonal expan-
sion, and subsequent terminal differentiation into adipocytes, as
well as a complex sequence of changes in gene expression and
1
diabetes, and cancer. The obese condition is generally aggravated
2
by excess accumulation of lipid and triglycerides in adipose tissue.
This adipose tissue is crucial metabolic organ that controls energy
homeostasis, through the regulation of both food intake and
energy expenditure. Obesity is known to be associated with an
excessive growth of adipose tissue mass by increasing the number
1
0
the storage of lipids. 3T3-L1 cells have been served as well estab-
lished in vitro assay system to assess adipogenesis and adipocyte
3
,4
11
and size of fat cells. The amount of adipose tissue mass can be
regulated by the suppression of adipogenesis, which is the devel-
differentiation.
During our search for a new regulator of adipocyte differentia-
tion from medicinal plants, the methanolic extract of Idesia poly-
carpa Maxim. fruits (Flacourtiaceae) showed significant inhibitory
activity on adipocyte differentiation (46.1 ± 4.9% of the control at
5
opment of mature fat cells from fibroblastic preadipocytes. Adipo-
kines, such as peroxisome proliferator activated receptor
and CCAAT/enhancer binding protein (C/EBP) directly exert influ-
ence on development of fat cell.6 Especially, PPAR
, highly
c (PPAR)
c
a concentration 100
oinflammatory effect of compounds isolated from I. polycarpa.
lg/mL). We previously reported the anti-neur-
12
expressed in adipose tissue, plays essential roles in adipogenesis
and glucose and lipid homeostasis, and is the predominant molec-
ular target for the insulin-sensitizing thiazolidinediones (TZD).
Sterol regulatory element binding protein 1c (SREBP1c) and its tar-
get genes, fatty acid synthase (FAS) and steroylcoenzyme A
Also, it was reported that idesolide, an isolate of I. polycarpa has
protective effect on skeletal muscle atrophy and inhibits the adipo-
genic differentiation of mesenchymal cells through the suppres-
sion of nitric oxide production. To date, there have been no
previous studies on the anti-adipogenic activity of I. polycarpa
using 3T3-L1 cells. In the course of our further attempt to afford
the anti-adipogenic constituents from I. polycarpa, we isolated
7
1
3
⇑
Corresponding author. Tel.: +82 2 880 7859; fax: +82 2 877 2933.
E-mail address: shsung@snu.ac.kr (S.H. Sung).
0
960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.011

M. Lee et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3170–3174
3171
ten compounds including to one new compound. In the present
study, we elucidated the anti-adipogenic activity of idescarpin
A
OH
HO
HO
6'
(1) through measuring gene expression of adipokines.
O
The I. polycarpa fruits were collected in the Nambu forest of
1'
OH O
O
7''
O
Seoul National University, Beagwoon Mountain, Gwangyang,
Jeollanam-do, Korea in Autumn 2010 and authenticated by Dr. Jong
Hee Park, professor of Pusan National University. A voucher speci-
men (CS76-1) has been deposited at the Herbarium of the Medici-
nal Plant Garden, College of Pharmacy, Seoul National University,
Koyang, Korea. The dried plant material (2.4 kg) was pulverized
and then extracted with 80% methanol (4 L, 3 h ꢀ 3) by ultrasoni-
cation at room temperature. The methanolic extract was concen-
trated in vacuo to give a crude extract (584 g). This methanolic
7
OH
6''
1
HO
O
1''
6
2
2''
1
B
control
25μM
50μM
extract was suspended in H
n-hexane (3 L), CHCl (3 L), EtOAc (3 L) and n-BuOH (3 L), giving a
solid residue of 24.7, 12.1, 119.3 and 109.5 g, respectively. The
CHCl , EtOAc and n-BuOH fractions showed significant inhibitory
effect on adipocyte differentiation (2.4 ± 4.4%, 8.7 ± 1.7%, and
3.7 ± 1.5% at a concentration 100 g/mL, respectively). To search
anti-adipogenic constituents, CHCl and n-BuOH fractions were
used for the follow-up isolation work with the exception of the
2
O and partitioned successively with
3
Fig. 2. Effects of idescarpin (1) on adipokine expression in 3T3-L. (A) Chemical
structure of idescarpin (1). (B) Confluent 3T3-L1 preadipocytes were differentiated
into adipocytes in a medium with different concentrations (25 and 50 lM) of
idescarpin (1). On 8 days, stained cells with Oil Red O were photographed with a
phase-contrast microscope (original magnification ꢀ 100). (C) On 4 and 8 day, total
RNAs were harvested and estimated by quantitative real time PCR analysis. The
3
2
l
3
previously reported EtOAc fraction.12 The CHCl
fraction (12.1 g)
was subjected to ODS gel column chromatography eluted with a
gradient mixture of H O–CH OH to yield 10 subfractions (C1–
C10). C2 was subjected to preparative HPLC (C18, 500 ꢀ 20 mm,
CH OH–H O, 40:60, 4 mL/min) to yield three subfractions
C2-1–C2-3). Compound 3 (113.7 mg) obtained from C2-3 by
recrystallization (CH OH). C4 yielded compound 6 (43.2 mg)
through semi-preparative HPLC (YMC J’Sphere, C18, 250 ꢀ 10 mm,
CH OH–H O, 43:57, 2 mL/min). C9 was further subjected to MPLC
RediSep silica gel, CHCl ? CH OH, 30 mL/min) to yield eight frac-
relative expression level of PPAR
determined after normalization to the GAPDH level. (D) Cell lysates were prepared
and the PPAR , C/EBP , and b-actin protein levels were detected by Western blot
c, C/EBPa, SREBP1, SCD-1, FAS, and aP2 was
3
c
a
2
3
analysis on 4 and 8 days. The relative expression levels of iNOS and COX-2 were
determined by densitometry and normalized by b-actin. Results represent the
mean ± SD of three independent experiments, each performed using triplicate
3
2
⁄
⁄⁄
⁄⁄⁄
28
wells. p <0.05, p <0.01, p < 0.001, compared with the differentiated cells.
(
3
Table 1
3
2
1
H and 13C NMR data and two-dimensional NMR correlations of compound 8
(
3
3
tions (C9-1–C9-8). Compound 4 (5.7 mg) and 5 (41.5 mg) yielded
Position
8
from C9-2 and C9-8, respectively, through recrystallization
1H (J = Hz)
13C
1
1
HMBC
H– H COSY
(
CH
HP-20 resin using CH
six subfractions (B1–B6). The B4 (21.2 g) was chromatographed on
silica gel column eluted by CHCl –CH OH–H O gradient and
yielded nine subfractions (B4-1–B4-9). B4-8 and B4-9 was further
3
OH). The n-BuOH fraction (109.5 g) was eluted from Diaion
1
2
3
4
5
6
7
145.1
151.4
121.2
127.6
118.1
134.6
61.6
3
OH–H O gradient as a mobile phase to afford
2
6.87 (1H, dd, 7.6, 1.2)
7.00 (1H, t, 7.8)
6.83 (1H, dd, 8.1, 1.4)
1, 2, 4, 5
1, 2, 3, 6
1, 2, 3, 4
4
3, 5
4
3
3
2
fractionated by preparative HPLC (C18, 500 ꢀ 20 mm, CH
3
OH–H
2
O,
4.81 (1H, d, 13.4)
1, 5, 6
3
5:65, 4 mL/min) to afford six subfractions (B4-8-1–B4-8-6) and
4.67 (1H, d, 13.6)
0
0
ten subfractions (B4-9-1–B4-9-10), respectivly. Compound
7
1
4.61 (1H, d, 7.9)
3.51 (1H, t, 8.6)
3.42 (1H, t, 9.0)
3.37 (1H, m)
3.55 (1H, dd, 7.1, 2.2)
3.76 (1H, dd, 11.1, 2.1)
3.90 (1H, dd, 11.1, 6.9)
4.84 (1H, d, 7.7)
3.33 (1H, d, 6.0)
3.65 (1H, m)
107.7
76.2
78.7
72.4
77.7
68.8
1
3 , 4
2 , 4
3 , 5 , 6
4 , 6
1
1 , 5
6
3 , 5 , 6
2
5 , 6
2 , 4
2
2
2
0
0
0
0
0
00
00
0
00
0
0
0
0
0
0
0
0
2
1 , 3
(
11.8 mg), compound 2 (112.5 mg), and compound 1 (3 g) were ob-
0
0
3
2 , 4
tained from B4-8-1, B4-8-2, and B4-8-5, respectively. Compound
0
0
0
4
3 , 5
1
5
0
(14.8 mg) was purified using preparative HPLC (C18
00 ꢀ 20 mm, CH OH–H O, 35:65, 4 mL/min) from B4-8-6, respec-
(18.5 mg). Compound
10.9 mg) was isolated through preparative HPLC (C18
,
0
0
0
5
4 , 6
0
0
3
2
6 -Ha
5
0
0
tively. B4-9-6 yielded compound
(
9
8
6 -Hb
0
0
0
0
00
1
2
3
4
5
6
100.9
72.7
74.5
74.6
75.8
63.4
2
,
00
00
00
00
00
00
00
1 , 3
5
00 ꢀ 20 mm, CH
3
OH–H
2
O, 30:70, 4 ml/min) from B4-9-10.
00
00
0
00
00
00
00
2 , 4
Compound 8 was obtained as yellowish syrup, and its molecular
00
00
00
3.37 (1H, m)
3.71 (1H, m)
3.67 (1H, d, 11.9)
3.83 (1H, dd, 9.9)
3 , 5
0
00
formula of C19
H
28
O
13 was established by the observed pseudomo-
4 , 6
ꢁ
00-Ha
00, 400
00
5
lecular ion peak at m/z 463.1454 [MꢁH] (calcd for C19
28 13
H O ,
0
0
00
63.1452) in the negative HRFABMS. The 1H NMR spectroscopic
data revealed signals for 1, 2, 6-trisubstituted aromatic ring
7.00 (1H, t, J = 7.8 Hz, H-4), 6.87 (1H, dd, J = 7.6, 1.2 Hz, H-3),
6 -Hb
4
[d
H
and 6.83 (1H, dd, J = 8.1, 1.4 Hz, H-5)], signals for one methylene
Table 2
Effects of the compounds isolated from I.polycarpa on adipocyte differentiation in
3T3-L1 preadipocytes
OH
Compound
IC50
(
l
M)
Compound
IC50 (lM)
6
''
O
HO
HO
1''
OH
1
2
3
4
5
6
23.2
43.0
34.8
118.8
>150
127.2
7
8
9
10
110.3
111.2
>150
66.8
O
6
'
OH
HO
HO
6
O
: HMBC correlation
1' O
OH
HO
1
2
: 1
1
H- H COS Y correlation
EGCG
112.0
8
IC50 means the 50% inhibitory concentration (
lM) on on adipocyte differentiation in
3T3-L1 preadipocytes. EGCG was used as the positive control.
Fig. 1. Structure and key 2D NMR correlations of compound 8.

3
172
M. Lee et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3170–3174
PPARγ
C/EBPα
SREBP1
C
1
1
1
1
1
0
0
0
0
0
.80
.60
.40
.20
.00
.80
.60
.40
.20
.00
1.40
1.20
.00
0.80
.60
0.40
.20
1
1
0
0
0
0
.20
.00
.80
.60
.40
.20
1
0
0
0.00
0.00
0
25
50
0
25
50
0
25
50
µM
µM
µM
SCD-1
FAS
aP2
1
1
0
0
0
0
0
.20
.00
.80
.60
.40
.20
.00
1.20
1.00
0.80
0.60
0.40
0.20
0.00
1.40
1
1
0
0
0
0
.20
.00
.80
.60
.40
.20
0.00
0
25
50
0
25
50
0
25
50
µM
µM
µM
4
day
8 day
.
4
day
8 day
D
5
5
7 kDa
4 kDa
PPARγ
C/EBPα
β-actin
42 kDa
3 kDa
4
Idescarpin (μM)
0
25
50
0
25
50
PPARγ
C/EBPα
1
1
1
0
0
0
0
0
.40
.20
.00
.80
.60
.40
.20
.00
1.20
1
0
0
0
0
.00
.80
.60
.40
.20
0.00
0
25
µM
50
0
25
µM
50
4
day
8 day
Fig. 2. (continued)

M. Lee et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3170–3174
3173
[
7
(
d
H
4.81 (1H, d, J = 13.4 Hz, H-7a) and 4.67 (1H, d, J = 13.6 Hz, H-
b)], signals for two anomeric protons of glucose moiety [d 4.61
H
1H, d, J = 7.9 Hz, H-1 ) and d 4.84 (1H, d, J = 7.7 Hz, H-1 )], which
25 and 50
l
M. Although PPAR
c
and C/EBP
a
are known as master
H
regulators of adipogenesis, this study showed conflicting results
(Fig. 2C and D). These results suggested that idescarpin (1) affects
the suppression of adipocyte differentiation, in spite of the activa-
0
00
were similar to those of compound 6 except for an additional
glucose moiety. Acid hydrolysis of compound 8 and HILIC–MS
analysis afforded
and mass spectra (m/z 160.83 [C
[
[
hydrolysis were compared with the authentic sample of
The coupling constant of doublet at d
(
The HMBC correlations of H-1 –C-1 and H-1 –C-6 indicated that
the two glucose units formed (1 ? 6) glycosidic linkage and were
located at C-1 of the aglycone (Fig. 1). Therefore, compound 8
tion of PPAR
c expression. SREBP1c regulates the expression of
D
-glucose.14 The retention time (t
R
7.02 min)
genes related to the metabolism of lipids and cholesterol and the
enzymes involved in lipogenesis and fatty acid desaturation. The
ꢁ
6
6
H
12
O
6
ꢁH
2
OꢁH] , 178.77
ꢁ
ꢁ
C
6
H
12
O
6
ꢁH] ,
224.79
[C
H
6 12
O
6
+HCOOHꢁH] ,
386.84
activation of SREBP1c promotes the expression of FAS and SCD-
ꢁ
8,27
2C
6
H
12
O
6
ꢁH
2
O+HCOOHꢁH] ) of the sugar released on acid
-glucose.
4.84 (J = 7.7) and d 4.61
J = 7.9) established the b-configuration of the glucose moiety.
1.
As shown in Figure 2C, the decrease in mRNA levels of
D
SREBP1c, SCD-1, and FAS by idescarpin (1) during adipocyte differ-
entiation also implies that the inhibition of adipocyte differentia-
tion was mediated by the regulation of lipogenesis. Idescarpin (1)
slightly reduced the gene expression of aP2, which plays central
roles in the linkage pathway of obesity to insulin resistance and
H
H
0
00
0
1
1
fatty acid metabolism.
was confirmed as 6-(oxymethyl)-2-hydroxyphenyl-O-b-
pyranosyl-(1 ? 6)-b- -glucopyranoside.
-(oxymethyl)-2-hydroxyphenyl-O-b-
-glucopyranoside (8) Yellowish syrup; ½
MeOH); IR (KBr) max: 3365, 2925, 1738, 1652, 1596, 1558, 1507,
473, 1365, 1296, 1202, 1034 cm ; R
D
-gluco-
In conclusion, it was demonstrated that methanolic extract of I.
polycarpa fruits and its isolated constituent affect the suppression
of adipogenesis in 3T3-L1 cells. In the course of the mechanismic
investigation of these natural products, we suggested that idescar-
pin (1), the most potent constituent of I. polycarpa can be the
potent therapeutic agent against obesity and diabetes though the
D
6
D
D-glucopyranosyl-(1 ? 6)
2
D
5
-
b-
a : +27.0 (c 0.2,
ꢂ
m
ꢁ
1
1
f
: 0.15 (Silica TLC, C/M/
W = 15:4:1), 0.86 (RP TLC, 40% M); H NMR (500 MHz, CD OD),
OD), see Table 1; HRFABMS
13, 463.1452).
1
3
anti-adipogenic activity and the up-regulation of PPARc expression.
1
3
see Table 1; C NMR (125 MHz, CD
3
ꢁ
m/z 463.1454 [MꢁH] (calcd for C19
28
H O
1
5
Acknowledgments
The nine known compounds were identified as idescarpin (1),
1
6
17
18
poliothrysin (2), (ꢁ)-idesolide (3), E-p-methylcoumarate (4),
1
2
18
19
This work was supported by the Global Leading Technology
Program (GLST-OSP project No. 10039303) of the Office of Strategic
R&D Planning (OSP) funded by the Ministry of Knowledge Econ-
omy, Republic of Korea.
idesolidine (5),
idesin (6),
salirepin (7),
hydrangeifolin I
2
0
21
(
9), itoside I (10), by comparing the measured spectroscopic
data with published values. To examine the effect of the com-
pounds (1–10) isolated from I. polycarpa on the differentiation of
preadipocytes into adipocytes, confluent 3T3-L1 preadipocytes
were treated with various concentrations of compounds during
differentiation (days 0–8).22 On day 8, the differentiated adipocytes
were stained with Oil Red O solution, and the lipid contents were
quantified spectrophotometrically at 544 nm, which enables the
analysis of the structure–activity relationship for these com-
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.04.
0
11.
2
3
pounds. Compounds 1–3 and 7–9 were found to significantly re-
duce lipid accumulation in 3T3-L1 preadipocytes, compared to
positive control, epigallocatechin-3-gallate (EGCG) (Table 2). Espe-
cially, compounds 1–3 with 1-hydroxy-6-oxo-2-cyclohexenecarb-
oxylate moiety exhibited more potent inhibitory activities on
adipocyte differentiation than the other compounds. In previous
Letter, idescarpin (1) and 1-hydroxy-6-oxo-2-cyclohexenecarboxy-
lic acid inhibited lipopolysaccharide-induced nitric oxide produc-
tion in BV2 microglia.12 Idesolide (3) also were reported to
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2
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1. Kong, C. S.; Kim, J. A.; Kim, S. K. Food Chem. Toxicol. 2009, 47, 2401.
12. Kim, S. H.; Jang, Y. P.; Sung, S. H.; Kim, Y. C. Plant Med. 2007, 73, 167.
1
2
1
1
3. Hwang, J. H.; Moon, S. A.; Lee, C. H.; Byun, M. R.; Kim, A. R.; Sung, M. K.; Park, H.
J.; Hwang, E. S.; Sung, S. H.; Hong, J. H. Eur. J. Pharmacol. 2012, 685, 218.
4. Compound 8 (2 mg) was dissolved in 6 N HCl and the solution was heated to
90 °C for 2 h. After neutralizing acidic solution with silver carbonate, the
(
1) showed the most potent inhibitory activity on adipocyte differ-
entiation with IC50 values of 23.2 M. As shown in Fig. 2B, fat drop-
let formation was decreased at concentrations of 25 and 50 M,
1
l
solvent was evaporated to dryness under
extracted with EtOAc and H O, successively, and the aqueous layer was
concentrated to dryness. The dried reactant was subjected to Hydrophilic
interaction liquid chromatography–electrospray ionisation-mass spectrometry
(HILIC–ESI-MS). The HPLC–ESI-MS system consisted of a Finnigan Surveyor
HPLC system with a pump, an autosampler and the Finnigan LCQ Advantage
2
N . The reaction mixture was
l
2
indicated by Oil Red O staining. The idescarpin (1) on gene and pro-
tein expression of adipokines was elucidated by real-time PCR and
Western blot analysis, respectively, compared with the fully differ-
entiated adipocytes without sample treatment at the gene level
Ò
with Xcalibur software. Separation was achieved at 30 °C on a ZIC -HILIC
2
4,25
(
Fig. 2C).
Adipocyte differentiation causes a series of pro-
and C/EBP
column (5 mm, 4.6 mm id ꢀ 100 mm, SeQuant AB). The mass spectrometer
grammed changes in specific gene expressions. PPAR
c
a
was operated with the ion source voltage set to ꢁ3000 V, capillary voltage
ꢁ
30 V, capillary temperature 300 °C, tube lens offset ꢁ60 V, sheath gas 40
are at the center of this network and oversee the entire terminal
(
arbitrary units) and auxiliary gas 30 (arbitrary units). The mobile solvent
differentiation process.26 Idescarpin (1) dramatically suppressed
consisted of (A) 0.03% formic acid in water and (B) 100% acetonitrile at a flow
rate of 0.3 mL/min, with isocratic conditions of 30% A and 70% B for 15 min.
5. Chou, C.; Lin, L.; Tsai, W.; Hsu, S.; Ho, L. J. Nat. Prod. 1997, 60, 375.
the induction of C/EBP
creased the induction of PPAR
a
expression, whereas it significantly in-
expression at concentrations of
1
c

3
174
M. Lee et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3170–3174
1
1
6. Gibbsons, S.; Gray, A. I.; Waterman, P. G. Nat. Prod. Lett. 1993, 3, 59.
7. Kim, S. H.; Sung, S. H.; Choi, S. Y.; Chung, Y. K.; Kim, J.; Kim, Y. C. Org. Lett. 2005,
the QIAGEN kits for SYBRÒ Green-based real time RT-PCR were PPAR
(NM_01146), C/EBP (NM_007678), SREBP1 (NM_011480), SCD-1
c
a
7
, 3275.
(NM_009127), FAS (NM_007988) and glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) (NM_008084) and were obtained from QIAGEN
Korea Ltd, Seoul, Korea. The amplification cycles were carried out at 95 °C for
20 s, 60 °C for 20 s, and 72 °C for 20 s. The last cycle was followed by a final
extension step at 72 °C for 5 min. Quantitative SYBR Green real-time PCR was
performed with an Applied Biosystems 7300 real-time PCR System (Life
Technologies Corporation, Carlsbad, CA, USA) and analyzed by means of
1
1
8. Ayer, W.; Trifonov, L. S. Phytochemistry 1995, 38, 371.
9. Ekabo, O. A.; Farnsworth, N. R.; Santisuk, T.; Reutrakul, V. Phytochemistry 1993,
3
2, 747.
0. De Tommasi, N.; Rastrelli, L.; Cumanda, J.; Speranza, G.; Pizza, C. Phytochemistry
996, 42, 163.
2
2
2
1
1. Chai, X. Y.; Xu, Z. R.; Ren, H. Y.; Shi, H. M.; Lu, Y. N.; Li, F. F.; Tu, P. F. Helv.. Chim.
Acta 2007, 90, 2176.
t
comparative C quantification (Popivanova et al., 2008). GAPDH was amplified
2. Mouse embryo fibroblasts 3T3-L1 cells were obtained from the American Type
Culture Collection (Manassas, VA, USA) and incubated in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% bovine calf serum until
confluence. Two days after confluence (designated day 0), preadipocytes were
stimulated to differentiate with differentiation medium [DM, DMEM with 10%
as an internal control. The C
values of the target genes (
t
values of GAPDH were subtracted from the C
). The values of the treated cells were
t
D
C
t
DC
t
compared with the
t
DC values of the untreated cells.
25. 3T3-L1 cells were washed twice with cold PBS, and then lysed in an ice-cold
RIPA buffer (Cell Signaling, Beverly, MA, USA) containing 0.5 mM DTT, 1 mM
PMSF, and 1% protease inhibitor cocktail. The lysate was centrifuged for 20 min
at 12,000ꢀg at 4 °C, and the supernatants were collected. Protein content was
determined by using the BCA protein assay (Pierce, Rockford, IL, USA). Total
Fetal Bovine Serum (FBS), 0.5 mM 3-isobutyl-1-methyl-xanthine, 10
insulin, and 1 M dexamethasone] for 2 d (day 2). Cells were then maintained
in DM (DMEM with 10% FBS and 10 g/mL insulin) for another 2 d (day 4),
followed by culturing with DM (DMEM with 10% FBS) for an additional 4 d (day
). All media contained 100 IU/mL penicillin and 100 g/mL streptomycin.
Cells were maintained at 37 °C in a humidified atmosphere of 95% air–5% CO
lg/mL
l
l
proteins (30 lg) were separated by 10% SDS–polyacrylamide gel
8
l
electrophoresis, transferred to polyvinylidene difluoride membranes
(Millipore, Billerica, MA, USA). After blocking in TBST (100 mM Tris, pH 8.0,
150 mM NaCl, and 0.1% Tween 20) with 5% non-fat dry milk for 1 h at room
temperature, the membrane was incubated overnight at 4 °C with 1:1000
2
.
The purity of each compound was verified as >95% by HPLC. Test compounds
were dissolved in DMSO (final concentration of 0.1% in media). The cultures
were treated with test samples for the whole culture period (day 0–8).
3. On day 8, lipid droplets in cells were stained with Oil Red O (ORO). Briefly,
culture dishes were washed three times with phosphate buffered saline (PBS)
and fixed with 10% formalin for 1 h at room temperature. After fixation, cells
were washed once with PBS and stained with a filtered ORO solution (6 parts of
saturated 0.6% ORO in isopropyl alcohol and 4 parts of water) for 15 min at
room temperature. Cells were washed twice with water for 15 min and
visualized. To quantify the intracellular lipids, spectrophotometrical
quantification of the stain was performed by dissolving the stained lipid
droplets with 4% Nonidet P-40 in isopropyl alcohol for 5 min. The absorbance
was measured at 544 nm.
diluted PPAR
Santa Cruz, CA, USA). After incubation with 1:2000 diluted horseradish
peroxidase-conjugated goat anti-rabbit immunoglobulin secondary
c and C/EBPa primary antibodies (Santa Cruz Biotechnology,
2
G
antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room
temperature, immunoreactive proteins were visualized by an enhanced
chemiluminescent
solution
(Amersham,
Uppsala,
Sweden).
After
normalization to b-actin, density values for the protein bands of interest
were expressed as a percentage of the control using ImageJ software (http://
rsweb.nih.gov/ij).
26. Farmer, S. R. Cell Metab. 2006, 4, 263.
27. Yao-Borengasser, A.; Rassouli, N.; Varma, V.; Bodles, A. M.; Rasouli, N.; Unal, R.;
Phanavanh, B.; Ranganathan, G.; McGehee, R. E.; Kern, P. A. J. Clin. Endocrinol.
Metab. 2008, 93, 4431.
2
4. The total RNA was extracted from the 3T3-L1 cells using RNease Plus Kit
(
QIAGEN Korea Ltd, Seoul, Korea). cDNA was synthesized with 1 lg of total
RNA using QuantiTech Reverse Transcription Kit (QIAGEN Korea Ltd., Seoul,
Korea), and then was mixed with QuantiFast SYBR Green PCR master mix
28. Statistical analysis was performed using Student’s t test. Data were expressed
as the means ± standard deviation (SD). Statistical significance was
represented with an asterisk for p values <0.05, with two asterisks for p
values <0.01 and three asterisks for p values <0.001.
(
QIAGEN Korea Ltd, Seoul, Korea) and specific primers. The conventional PCR
products were generated by AccuPower PCR Premix (Bioneer Corp., Daejeon,
Korea) in a total reaction volume of 20 L. The PCR specific primers used with
l