Chemical constituents of Triticum aestivum and their effects on adipogenic differentiation of 3T3-L1 preadipocytes

Abstract

In this report, we investigated the anti-obesity effect of wheat sprouts and their component compounds. Twenty compounds (1–20) were isolated from Triticum aestivum. Among them, glycolipids 1–5 were determined for the first time from T. aestivum and its sprouts. The HPLC analysis demonstrated that compounds 1–3, 5, 8, 12, and 14 were major peak in the HPLC chromatogram of the active fraction. The effects of the compounds on lipid accumulation were assessed at concentrations ranging from 1.0 to 100 lM. At concentration of 10.0 lM, compounds 1–7, 10-15, and 17–19 significantly decreased lipid accumulation in 3T3-L1 preadipocytes. Glycolipids 1, 2, and phenolic 17 significantly reduced lipid accumulation in the differentiated adipocytes in a concentration-dependent manner. Quantitative analysis based on measurement of the optical density of Oil Red O indicated that, at 100 lM, compounds 1, 2, and 17 reduced lipid accumulation by 41, 37, and 48 %, respectively, compared with the positive control.

Full text

RESEARCH ARTICLE
Chemical constituents of Triticum aestivum and their effects
on adipogenic differentiation of 3T3-L1 preadipocytes
Bui Thi Thuy Luyen •Nguyen Phuong Thao •
Bui Huu Tai •Ji Young Lim •Hyeon Hui Ki •
Dae Ki Kim •Young Mi Lee •Young Ho Kim
Received: 25 July 2014 / Accepted: 28 August 2014 / Published online: 23 September 2014
ÓThe Pharmaceutical Society of Korea 2014
Abstract In this report, we investigated the anti-obesity
effect of wheat sprouts and their component compounds.
Twenty compounds (1–20) were isolated from Triticum
aestivum. Among them, glycolipids 1–5were determined
for the first time from T. aestivum and its sprouts. The
HPLC analysis demonstrated that compounds 1–3,5,8,12,
and 14 were major peak in the HPLC chromatogram of the
active fraction. The effects of the compounds on lipid
accumulation were assessed at concentrations ranging from
1.0 to 100 lM. At concentration of 10.0 lM, compounds
1–7,10-15, and 17–19 significantly decreased lipid
accumulation in 3T3-L1 preadipocytes. Glycolipids 1,2,
and phenolic 17 significantly reduced lipid accumulation in
the differentiated adipocytes in a concentration-dependent
manner. Quantitative analysis based on measurement of the
optical density of Oil Red O indicated that, at 100 lM,
compounds 1,2, and 17 reduced lipid accumulation by 41,
37, and 48 %, respectively, compared with the positive
control.
Keywords Triticum aestivum Wheat sprout 
Glycolipid 3T3-L1 preadipocytes Anti-obesity
Introduction
Obesity is a global pandemic health threat not only in
developed countries but also increasingly in low- and
middle- income countries. The World Health Organization
estimates that approximately 2.3 billion adults will be
overweight and more than 700 million will be obese by
2015 (George et al. 2014). Obesity is characterized by
excess adipose mass and adipose tissue expansion, which
result from increased numbers and sizes of adipocytes. It
has been associated with both metabolic and chronic dis-
eases such as type 2 diabetes, heart diseases, inflammation,
hypertension, and several forms of cancer. Currently, there
are various methods of preventing/treating obesity includ-
ing dietary interventions, exercise, behavioral therapies,
and medications (Villareal et al. 2011). Various anti-
obesity drugs including orlistat, sibutramine, lorcaserin,
phendimetrazine, and diethylpropion have been developed
and approved by the Food and Drug Administration
(Padwal and Majumdar 2007; George et al. 2014). How-
ever, they have been reported to have several serious
adverse effects in clinical practice, including headache,
vomiting, gastrointestinal adverse effects, and heart attack
(Sayin and Guldal 2005; Padwal and Majumdar 2007). As
a result, natural products from edible plants have attracted
interest for discovery of new anti-obesity agents that are
cheap and highly compatible with dietary intake, and have
no harmful effects on the body.
B. T. T. Luyen N. P. Thao B. H. Tai Y. H. Kim (&)
College of Pharmacy, Chungnam National University,
Daejeon 305-764, Republic of Korea
e-mail: yhk@cnu.ac.kr
N. P. Thao B. H. Tai
Institute of Marine Biochemistry (IMBC), Vietnam Academy of
Science and Technology (VAST), 18 Hoang Quoc Viet,
Caugiay, Hanoi, Vietnam
J. Y. Lim Y. M. Lee (&)
Department of Oriental Pharmacy, College of Pharmacy,
Wonkwang University and Wonkwang Oriental Medicines
Research Institute, Iksan, Jonbuk 570-749, Republic of Korea
e-mail: ymlee@wku.ac.kr
H. H. Ki D. K. Kim
Department of Immunolgy and Institute of Medical Sciences,
Medical School, Chonbuk National University, Jeonju,
Jeonbuk 561-756, Republic of Korea
123
Arch. Pharm. Res. (2015) 38:1011–1018
DOI 10.1007/s12272-014-0478-2

Triticum aestivum (TA, wheat) is an excellent food
source and can be harvested on a large scale. It is cultivated
primarily in temperate regions and accounts for more than
one third of global cereal production. TA products, notably
wheat germ, wheat bran, and wheat grass juice, have sev-
eral pharmacological properties, including anti-cancer,
anti-colitis, anti-inflammation, and antioxidant activities
(Pesarini et al. 2013). Our previous studies indicate that
wheat grass extract produces excellent anti-obesity effects
by inhibiting weight gain caused by a high-fat diet and by
lowering neutral lipid levels in the blood in a high-fat-diet-
induced obesity animal model (Lee et al. 2013). A 50 %
ethanol extract of TA sprouts and its dichloromethane
fraction produced potent anti-adipogenic effects by inhib-
iting the transactivation of PPARc(Lee et al. 2011). In the
present study, we aimed to identify chemical constituents
of TA sprouts and to evaluate their effects on adipogenic
differentiation in 3T3-L1 preadipocytes.
Materials and methods
General experimental procedures
The UV spectra were acquired using a JASCO V-550 UV/
VIS spectrometer. FT-IR spectra were recorded on a
JASCO Report 100 infrared spectrophotometer. The NMR
spectra were measured using a JEOL ECA 600 spectrom-
eter (JEOL, Tokyo, Japan) with TMS as the internal stan-
dard. The electronspray ionization (ESI) mass spectra were
performed on an AGILENT 1100 LC-MSD trap spec-
trometer (Agilent Technologies, Palo Alto, CA, USA). The
HR-ESI-MS were obtained from an Agilent 6530 Accu-
rate-Mass Q-TOF LC/MS system. Gas chromatography
spectra were recorded on a Shidmazu-2010 spectrometer
(Shimadzu, Kyoto, Japan). Silica gel (70–230, 230–400
mesh, Merck, Whitehouse Station, NJ), YMC RP-18 resins
(75 lm, Fuji Silysia Chemical Ltd., Kasugai, Japan) were
used as absorbents in the column chromatography. Thin
layer chromatography (TLC) plates (silica gel 60 F
254
and
RP-18 F
254
, 0.25 lm, Merck) were purchased from Merck
KGaA (Darmstadt, Germany). Spots were detected under
UV radiation (254 and 365 nm) and by spraying the plates
with 10 % H
2
SO
4
followed by heating with a heat gun.
Chemical reagents and standard compounds were pur-
chased from Sigma-Aldrich.
Plant materials
Triticum aestivum sprouts were purchased from Woori-
Milsak Company and National Institute of Crop Science
(Suwon, Korea). They were germinated and cultivated on
organic sterile peat-moss. After germination, TA was
cultivated for 2 weeks and then harvested, freeze-dried,
powdered and stored at 4 °C in the dark until use. A
voucher specimen (No. TA-10-01) was deposited at the
Herbarium of the College of Pharmacy, Chungnam
National University, Daejeon 305-764, Republic of Korea.
Extraction and isolation
Dried and powdered T. aestivum sprouts (1.0 kg) were
extracted with methanol under reflux conditions three times
(5 h each). The solvent was removed in a vacuum to give a
dark solid methanol extract (150.0 g). The crude extract was
suspended in 2 L of water and successively partitioned with
n-hexane, dichloromethane (CH
2
Cl
2
), ethyl acetate
(EtOAc), and n-butanol (BuOH) to give a hexane extract
(70.0 g), a CH
2
Cl
2
extract (6.0 g), an EtOAc extract (4.0 g),
and a BuOH extract (40.0 g) plus a water layer, respectively.
The hexane and dichloromethane extracts were combined
and fractionated by silica gel column chromatography (CC)
eluting with a gradient solvent system of hexane/acetone
(0–100 % volume of acetone) to obtain six fractions (HD-1
to HD-6). Fraction HD-3 (6.0 g) was subjected to silica gel
CC and eluted with n-hexane/CHCl
3
/MeOH (3/2/0.1, v/v/v)
to yield five smaller fractions (HD-3A to HD-3E). Fraction
HD-3A was separated by silica gel CC using n-hexane/
acetone (10/1, v/v) as the eluent and was further purified by
silica gel CC. Elution with n-hexane/EtOAc (20/1, v/v)
yielded compound 6(6.8 mg). Fraction HD-3D was
repeatedly chromatographed on a silica gel column and
eluted with n-hexane/acetone (10/1, v/v) to obtain com-
pound 8(50.0 mg). Fraction HD-6 (27.0 g) was further
separated by silica gel CC eluting with a gradient solvent
system of CH
2
Cl
2
/MeOH (0–100 % volume of MeOH) to
obtain six fractions (HD-6A to HD-6G). Fraction HD-6B
(6.0 g) was subjected to silica gel CC and was eluted with n-
hexane/acetone (3/1, v/v) to give four smaller fractions (HD-
6B1 to HD-6B4). Compounds 1(12 mg) and 4(140 mg)
were purified from fraction HD-6B1 by YMC CC using
acetone/H
2
O (8/1, v/v) as the eluent. Purification of fraction
HD-6B2 by YMC CC and elution with MeOH/acetone/H
2
O
(10/3/0.5, v/v/v) afforded compound 5(93 mg). Fraction
HD-6C (6.0 g) was subjected to silica gel CC, followed by
elution with CH
2
Cl
2
/acetone (2/1, v/v) and further purifi-
cation by YMC CC with a solvent system of MeOH/acetone/
H
2
O (10/1/0.1, v/v/v) obtained compounds 9(16 mg), 3
(98.0 mg) and 2(17 mg). Fraction HD-6E (3.0 g) was
fractioned by silica gel CC and eluted with CH
2
Cl
2
/n-hex-
ane/MeOH (2/3/0.1, v/v/v). It was then purified by YMC CC
with MeOH/acetone/H
2
O (10/1/1, v/v/v) as the eluent, to
afford compounds 7(200.0 mg), 10 (40.0 mg) and 11
(60.0 mg). Compounds 12–20 were obtained as described
previously (Luyen et al. 2014). The purity of the isolated
compounds was over 95 % by HPLC analysis.
1012 B. T. T. Luyen et al.
123

2-a-linolenoylglycerol 1-O-b-D-galactopyranoside (1)
Colorless wax;
1
H-NMR (400 MHz, CDCl
3
): 5.34 (6H,
overlapped, H-900,10
00,12
00,13
00,15
00,16
00), 2.80 (4H, t,
J=6.0 Hz, H-1100 ,14
00), 2.32 (2H, t, J=7.2 Hz, H-200),
2.05 (4H, overlapped, H-800 ,17
00), 1.59 (2H, br s, H-300 ), 1.29
(8H, overlapped, H-400,5
00,6
00,7
00), 0.97 (3H, t, J=7.6 Hz,
H-1800);
13
C-NMR (100 MHz, CDCl
3
): 174.0 (C-100), 132.0
(C-1600), 130.3 (C-1500 ), 128.4 (C-1300), 128.3 (C-1200 ), 127.8
(C-1000), 127.2 (C-900 ), 103.9 (C-10), 74.5 (C-50), 73.3 (C-30),
72.7 (C-3), 71.1 (C-20), 68.9 (C-40), 68.0 (C-2), 61.3 (C-1),
61.2 (C-60), 34.2 (C-200), 29.6 (C-400 ), 29.6 (C-500 ), 29.2 (C-
600), 29.1 (C-700 ), 27.2 (C-800 ), 25.5 (C-1100), 25.5 (C-1400 ),
24.8 (C-300), 20.5 (C-1700 ), 14.2 (C-1800 ).
3-a-linolenoylglycerol 1-O-b-D-galactopyranoside (2)
Colorless wax;
1
H-NMR (400 MHz, CDCl
3
): 5.33 (6H,
overlapped, H-900,10
00,12
00,13
00,15
00,16
00), 2.79 (4H, t,
J=6.0 Hz, H-1100 ,14
00), 2.32 (2H, t, J=7.2 Hz, H-200),
2.05 (4H, overlapped, H-800 ,17
00), 1.59 (2H, br s, H-300 ), 1.30
(8H, overlapped, H-400,5
00,6
00,7
00), 0.97 (3H, t, J=7.6 Hz,
H-1800);
13
C-NMR (100 MHz, CDCl
3
): 174.3 (C-100), 132.0
(C-1600), 130.2 (C-1500 ), 128.4 (C-1300), 128.2 (C-1200 ), 127.8
(C-1000), 127.1 (C-900 ), 103.5 (C-10), 74.4 (C-50), 73.3 (C-30),
71.1 (C-3), 71.4 (C-20), 68.9 (C-40), 68.5 (C-2), 65.0 (C-1),
61.1 (C-60), 34.0 (C-200), 29.6 (C-400 ), 29.6 (C-500 ), 29.2 (C-
600), 29.1 (C-700 ), 27.2 (C-800 ), 25.5 (C-1100), 25.4 (C-1400 ),
24.8 (C-300), 20.5 (C-1700 ), 14.2 (C-1800 ).
Triticumoside (17)
Pale yellow amorphous powder;
1
H-NMR (600 MHz,
CD
3
OD-d
4
): 7.29 (2H, s, H-20,6
0), 6.75 (1H, s, H-3), 6.46
(1H, br s, H-8), 6.19 (1H, br s, H-6), 6.13 (1H, d, J=3.0 Hz,
H-20’), 5.56 (1H, d, J=3.0 Hz, H-600 ), 4.98 (1H, d,
J=7.8 Hz, H-100 0 ), 3.85 (6H, s, 30,5
0-OCH
3
), 3.81 (3H, s,
300-OCH
3
), 3.80 (1H, dd, J=2.4; 12.0 Hz, H-6b00 0 ), 3.67
(1H, dd, J=5.4; 12.0 Hz, H-6a00 0 ), 3.49 (1H, t, J=7.8 Hz,
H-2000 ), 3.40 (2H, overlapped, H-3000 ,4
000 ), 3.25 (1H, m,
H-5000 );
13
C-NMR (150 MHz, CD
3
OD-d
4
): 183.8 (C-4),
166.4 (C-7), 164.9 (C-2), 163.3 (C-5), 159.5 (C-9), 155.6 (C-
100), 155.1 (C-300 ), 155.0 (C-30,5
0), 152.9 (C-500), 136.1 (C-40),
130.2 (C-10), 129.1 (C-400), 106.4 (C-3), 106.0 (C-1000 ), 105.5
(C-10), 105.0 (C-20,6
0), 100.4 (C-6), 95.4 (C-200), 95.3 (C-8),
95.0 (C-600), 78.3 (C-500 0 ), 77.8 (C-3000 ), 75.8 (C-200 0 ), 71.4 (C-
4000 ), 62.8 (C-600 0 ), 57.0 (30,5
0-OCH
3
), 56.9 (300-OCH
3
).
HPLC analysis condition
Samples were performed HPLC analysis using Waters
ACQUITY UPLC system associated with Photodiode
Array (PDA) detector. Running conditions were used as
following column Waters ACQUITY UPLC-BEH-C18
(2.1 950 mm, 1.7 lM), column temperature 30 °C,
wavelength 190–400 nm, gradient solvent system of A:
0.1 % H
3
PO
4
in water and B: 0.1 % H
3
PO
4
in acetonitrile
[time (% B): 0 (3 % B), 8.0 (25 % B), 10.0 (25 % B), 15.0
(70 %B), 17.0 (70 % B), 22.0 (100 % B), and 28.0 (100 %
B)], injection volume 2.0 lL, flow rate 0.6 mL/min.
Cell culture and adipocyte differentiation assay
3T3-L1 preadipocytes were obtained from American Type
Culture Collection (ATCC CL-173
TM
, Manassas, VA). The
cells were maintained with high-glucose Dulbecco’s
modified Eagle’s medium (DMEM) containing 10 % fetal
bovine serum (FBS), penicillin (100 U/mL) and strepto-
mycin (100 mg/mL) at 37 °C in a humidified CO
2
incu-
bator (5 % CO
2
, 95 % air). Passages 3 through 9 of the
cells were used in all experiments. Adipogenesis of pre-
adipocytes were stimulated by incubation with differenti-
ating medium (MDI) containing 0.5 mM of IBMX, 1.0 lM
of dexamethasone and 10 lg/mL of insulin for 2 days.
Then, culture medium was changed to DMEM/10 % FCS
containing insulin. After 2 days, the medium was replaced
with DMEM/10 % FCS and incubated for another 4 days
(8 days in total) in complete medium for fully differenti-
ation. To study the effects of and the compounds isolated
from T. aestivums extract on adipogenic differentiation, the
individual compounds were added until the time when the
cells were harvested for the assays.
Cell viability
The viability of 3T3-L1 preadipocytes were determined by
measuring the mitochondrial enzyme activity according to
the colorimetric MTT method. In brief, 3T3-L1 preadipo-
cytes were initially seeded at a density of 10
4
cells/well in
48-well plates and cultured for confluence. They were then
incubated in a complete medium containing various con-
centrations of compounds (1–100 lM). After 48 h, cells
were incubated with MTT solution (0.5 mg/mL) for 1 h at
37 °C. Formation of a violet precipitate, formazan, was
monitored in DMSO at a wavelength of 540 nm using a
microplate reader (Bio-Rad microreader, USA).
Oil Red O staining
On day 8, lipid droplets in cells were stained with Oil Red
O (Kasturi and Joshi 1982). Cell monolayers were washed
twice with phosphate buffered saline (PBS, pH 7.4) and
fixed with 3.7 % formaldehyde for 10 min. Fixed cell were
stained with 0.2 % Oil Red O-isopropanol for 1 h and
excess of stain was washed by 70 % ethanol and water.
Cell were then photographed using phase contract
Chemical constituents of Triticum aestivum and their effects 1013
123

microscopy. To quantify the intracellular lipids, dissolving
the stained oil droplets with isopropanol was performed
spectrophotometrical quantification at 510 nm. The results
were represented as a relative percentage of lipid accu-
mulation in cells versus MDI-treated positive control.
Statistical analysis
All experiment was performed repeatedly at least three
times. Values are expressed as mean ±SD. Statistical
analyses were performed by oneway ANOVA analysis
using Graph Pad software, P\0.05 and P\0.005 versus
control.
Results and discussion
Wheat sprouts of 3–4 cm length are usually obtained after
germination for 1 week. They were reported to have poten-
tial antioxidant activity (Falcioni et al. 2002; Calzuola et al.
2004), antimutagenic effects against bezo[a]pyrene (BP)-
induced mutagenicity, and reduced formation of BP metab-
olites by hepatic microsomes in BP- or phenobarbital-treated
rats (Peryt et al. 1992). Recently, c-aminobutyric acid and
ferulic acid were determined to be constituents of wheat
sprouts with activities against a-amylase (IC
50
5.4 ±0.2 and
9.5 ±0.1 mM, respectively) and a-glucosidase (IC
50
1.4 ±
0.4 and 4.9 ±0.3 mM, respectively) (Jeong et al. 2012). In
continuing research on the human nutrition and health ben-
efits of wheat sprouts, their extracts and fractions were
screened for anti-obesity effects. The results indicated that a
50 % ethanol extract as well as its dichloromethane fraction
exhibited potential inhibition of lipid accumulation during
differentiation of 3T3-L1 preadipocytes. In addition, wes-
tern blot analysis suggested that the potent anti-adipogenic
effects of the wheat sprout extract were due to inhibition of
PPARctransactivation. The literatures indicates that wheat
has nutrition and health benefits because it is rich in dietary
fiber, starches, minerals, phenolic compounds and other
phytochemicals. However, to date, few studies have focused
H OR2
O
OR1
O
OH
HO
OH
OR3
R1R2R3
1 H Len H
2 Len H H
3 Len Len H
4 Len Len Len
5 Len Len Gal
H OR
OR
OR
R
6 Len
7 Lin
OH
O
nm
n m
10 7 7
11 4 10
OR
O
7
R
8H
9 CH3
RO
R
12 H
13 Glc
14 6-LenGlc
Glc
O
O
HO
OH
OH
OR
R
18 H
19 CH3
N
OOGlc
H3CO
OCH3
O
OHO
OH O
OCH3
OCH3
OCH3
OH
OGlc
O
O
HO
17
15
20
N
N
N
N
NH2
O
OH OH
HO
16
Fig. 1 The structures of compounds isolated from Triticum aestivum.Len a-linolenoyl, Lin linoleoyl, Glc glucosyl, Gal galactosyl
1014 B. T. T. Luyen et al.
123

on the isolation and purification of bioactive constituents
from wheat sprouts. To clarify the chemical constituents of
wheat sprouts, dried and milled raw samples were macerated
and repeatedly extracted with methanol. The crude extract
was then partitioned in various solvents. Chemical constit-
uents were separated and purified by a combination of var-
ious column chromatographic techniques. Based upon the
results of TLC and NMR analysis, twenty compounds were
isolated from wheat sprouts. Their structures were deter-
mined by NMR, MS analysis, and comparison with the
published literatures. Briefly, the following isolated com-
pounds were identified (Fig. 1) including five glycolipids:
2-a-linolenoylglycerol 1-O-b-D-galactopyranoside (1) (Ha
et al. 2006), 3-a-linolenoylglycerol 1-O-b-D-galactopyran-
oside (2) (Tuntiwachwuttikul et al. 2004), 2,3-di-a-linole-
noylglycerol 1-O-b-D-galactopyranoside (3) (Ha et al. 2006),
2,3-di-a-linolenoylglycerol 1-O-(6-O-linolenoyl-b-D-galac-
topyranoside) (4) (Ruberto and Tringali 2004), and 2,3-di-a-
linolenoylglycerol 1-O-[a-D-galactopyranosyl-(1 ?6)-O-
b-D-galactopyranoside] (5) (Wegner et al. 2000); two gly-
cerides: trilinolenin (6) (Hui et al. 2003), trilinolein (7) (Park
et al. 2001); four unsaturated fatty acids: a-linolenic (8)
(Takaya et al. 2003), a-linolenic acid methyl ester (9) (Ta-
kaya et al. 2003), oleic acid (10) (Batchelor et al. 1974), and
12(Z)-octadecenoic acid (11) (Batchelor et al. 1974); three
sterol: b-sitosterol (12), daucosterol (13), 60-O-linolenly-
daucosterol (14) (Luyen et al. 2014); two alkaloids: (2R)-2-
O-b-D-glucopyranosyl-4,7-dimethoxy-2H-1,4-benzoxazin-
3(4H)-one (15), adenosine (16) (Luyen et al. 2014); and four
phenolics: triticumoside (17), isoorientin (18), isoscoparin
(19), and a-tocopherol (20) (Luyen et al. 2014). Among
them, compound 17 was determined to be a new compound,
Fig. 2 HPLC profile of 50 %
EtOH extract (A)ofTriticum
aestivum and its active CH
2
Cl
2
fraction (B)
Chemical constituents of Triticum aestivum and their effects 1015
123

named as triticumoside (Luyen et al. 2014). Compounds 6–
13,15,16, and 18–20 are found in different parts of wheat
such as seeds/flour, germ, bran, and leaves. However, they
have not been described previously in wheat sprouts. Gly-
colipids 1–5were isolated for the first time from T. aestivum
and its sprouts. To our knowledge, this is the most extensive
study on the chemical constituents of wheat sprouts. As in
our previous study, the 50 % ethanol extract and its dichlo-
romethane fraction showed marked inhibition of adipogenic
differentiation in 3T3-L1 preadipocytes. Thus, the 50 %
ethanol extract and dichloromethane fraction were subjected
to HPLC profiling and in comparison with each isolated
compound (Fig. 2). The HPLC profile demonstrated that
compounds 1–3,5,8,12, and 14 were major peak in the
HPLC chromatogram of the active fraction. Next, we
examined the effects of the isolated compounds on adipo-
genic differentiation of 3T3-L1 preadipocytes. First, the
cytotoxicity of each compound in 3T3-L1 cells was tested by
MTT assays. At the highest concentration (100 lM), the
compounds showed no significant effects on cell viability.
Thus, the effects of the compounds on lipid accumulation
were assessed at concentrations ranging from 1.0 to 100 lM.
The results indicate that, at a low concentration (1.0 lM),
there was little difference in lipid accumulation compared
with the control. However, at 10.0 lM, all of the compounds,
with exceptions of compounds 8,9,16, and 20 significantly
decreased lipid accumulation in 3T3-L1 preadipocytes
(Fig. 3). Glycolipids 1–5displayed potent inhibitory activity
at a concentration of 50.0 lM. However, with the exceptions
of compounds 1and 2, they did not showed dose-dependent
activity at high concentrations. Their activities did not differ
markedly at concentrations higher than 50.0 lM. Thus, the
optimal concentrations and mechanisms of action of these
compounds must be determined. To date, this is the first
report of the anti-obesity effects of glycolipids 1–5. In par-
ticular, glycolipids 1and 2significantly reduced lipid
accumulation in the differentiated adipocytes in a concen-
tration-dependent manner (Fig. 4).
Quantitative analysis based on measurement of the
optical density of Oil Red O indicated that, at 100 lM,
compounds 1and 2reduced lipid accumulation by 41 and
37 %, respectively, compared with the positive control
(PC). Sterol compounds 12–14 also exhibited inhibitory
activity at 10 lM, but this activity was not dose-dependent.
Of the alkaloids (compounds 15 and 16), the 1,4-benzox-
azinone derivative (15) was previously isolated from sev-
eral cereals such as wheat, corn, and rye. Benzoxazinones
are major compounds in poaceous plants and are involved
in the natural defense response of these plants against
insects (Oikawa et al. 2004). Recently, they have been
shown to have potential health-promoting anticancer, anti-
allergy and anti-inflammation effects. Thus, the anti-adi-
pogenic activity of compound 15 will support ongoing
efforts to utilize benzoxazinoids in health-promoting food
products. Phenolic compounds from wheat are expected to
be responsible for several pharmacological effects of wheat
NC
PC
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
0
20
40
60
80
100
120
Relative Optical density of ORO
at 510 nm (%)
**
*
**
*
*****
**
*
*
Compounds
Fig. 3 The effects of the compounds isolated from Triticum aestivum
on lipid accumulation in 3T3-L1 preadipocytes. All compounds were
pretreated at concentration of 10.0 lM. Data are presented as
mean ±SEM (*P\0.05, **P\0.005 vs PC). NC negative control,
PC MDI-treated positive control
1016 B. T. T. Luyen et al.
123

samples, such as antioxidant, anti-inflammation, a-gluco-
sidase, and anti a-amylase activities. In our experiments,
other than a-tocopherol (20), the phenolic compounds
(17–19) at 10.0 lM significantly reduced lipid accumula-
tion and showed dose-dependent inhibitory effects under
the in tested conditions. Isoorientin (18) and isoscoparin
(19) are luteolin C-glucosides, which are common, natu-
rally occurring C-glycosyl flavones in many plant species
(Yuan et al. 2014). These compounds have been reported to
have various biological properties, such as antioxidant
(Yuan et al. 2013; Deepha et al. 2014), anti-nociceptive
(Kuepeli et al. 2004), anti-diabetic (Alonso-Castro et al.
2012), and anti-inflammatory activities (Luyen et al. 2014).
Aspalathus linearis extract was reported to inhibit intra-
cellular lipid accumulation, and isoorientin was found to be
a major phenolic compound in A. linearis (Sanderson et al.
2014). However, no anti-adipogenic effects of isoorientin
have been reported to date. Triticumoside (17), an inter-
esting hybrid structure of flavonoid and polyphenol, was
newly identified in a natural source. It showed the highest
inhibitory activity among the phenolics (17–19). Cells
treated with compound 17 (1–100 lM) displayed signifi-
cant inhibition of lipid accumulation compared with the PC
(P\0.005) (Fig. 4). Quantitative analysis of intracellular
lipid accumulation indicated that, at 100 lM, compound 17
reduced lipid accumulation by 48 % compared with the
PC.
In summary, HPLC profiles and bioassay results indi-
cate that glycolipids, notably compounds 1–3, and 5are
major active components of a 50 % ethanol extract of TA
sprouts and its dichloromethane fraction, and inhibit adi-
pogenic differentiation of 3T3-L1 preadipocytes. A ben-
zoxazinone derivative (15) and phenolic compounds (17–
19) at concentration of 10.0 lM also significantly reduced
lipid accumulation in differentiated 3T3-L1 cells. Our
study is the first report of the anti-obesity effect of wheat
sprouts and their component compounds. The results
support the nutrition and health benefits of wheat sprouts
and their use as active constituents in health-promoting
food products.
NT
PC
1
10
0
20
40
60
80
100
120
Relative Optical density of ORO
at 510 nm (%)
**
**
Comp 2
B
NT
PC
1
10
100
0
20
40
60
80
100
120
Relative Optical density of ORO
at 510 nm (%)
Comp 17
C
**
**
NT
PC
1
10
100
100
0
20
40
60
80
100
120
Relative Optical density of ORO
at 510 nm (%)
*
**
Comp 1
Concentration (µM)
Concentration (µM)
Concentration (µM)
A
Fig. 4 The effect of three compounds 1(A), 2(B), and 17 (C) on the
lipid accumulation of adipocyte in a dose-dependent manner. 3T3-L1
preadipocytes were differentiated in the presence of various concen-
trations (0–100 lM) of the compounds. These cells were subjected to
determination of intracellular lipid accumulation by optical density of
Oil Red O (510 nm). Data are presented as mean ±SEM (*P\0.05,
**P\0.005 vs PC). NT negative control, PC MDI-treated positive
control
Chemical constituents of Triticum aestivum and their effects 1017
123

Acknowledgments This research was financially supported by the
Ministry of Knowledge Economy (MKE), Korea Institute for
Advancement of Technology (KIAT) through the Inter-ER Cooper-
ation Projects (R0002019) and the Priority Research Center Program
through the National Research Foundation of Korea (NRF) funded by
the Ministry of Education, Science, and Technology (2009-0093815),
Republic of Korea.
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