Six new chalcones from Angelica keiskei inducing adiponectin production in 3T3-L1 adipocytes

Article abstract of DOI:10.1271/bbb.110976

Angelica keiskei (Ashitaba in Japanese), a traditional herb in Japan, contains abundant prenylated chalcones. It has been reported that the chalcones from A. keiskei showed such bioactivities as anti-bacterial, anti-cancer and anti-diabetic effects. Xanth

Full text of DOI:10.1271/bbb.110976

Biosci. Biotechnol. Biochem., 76 (5), 961–966, 2012
Six New Chalcones from Angelica keiskei Inducing Adiponectin Production
in 3T3-L1 Adipocytes
y
Hiromu OHNOGI,1;2; Yoko KUDO,2 Kenichi TAHARA,2 Katsumi SUGIYAMA,2 Tatsuji ENOKI,2
Shoko HAYAMI,2 Hiroaki SAGAWA,2 Yuko TANIMURA,1 Wataru AOI,1;3 Yuji NAITO,1
1
Ikunoshin KATO,2 and Toshikazu YOSHIKAWA
1Department of Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine,
Kamigyo-ku, Kyoto 602-8566, Japan
2Takara Bio Inc., 3-4-1 Seta, Otsu, Shiga 520-2193, Japan
3Laboratory of Health Science, Kyoto Prefectural University, Sakyo-ku, Kyoto 606-8522, Japan
Received December 21, 2011; Accepted January 16, 2012; Online Publication, May 7, 2012
[doi:10.1271/bbb.110976]
Angelica keiskei (Ashitaba in Japanese), a traditional
herb in Japan, contains abundant prenylated chalcones.
It has been reported that the chalcones from A. keiskei
showed such bioactivities as anti-bacterial, anti-cancer
and anti-diabetic effects. Xanthoangelol, 4-hydroxyder-
ricin and six new chalcones were isolated in this study
from an ethanol extract of A. keiskei by octadecyl silyl
(ODS) and silica gel chromatography, and identified by
1D- and 2D-nuclear magnetic resonance (NMR) and
high-resolution mass spectrometric analyses. The chal-
cones from A. keiskei markedly increased the expression
of the adiponectin gene and the production of adipo-
nectin in 3T3-L1 adipocytes. These results suggest that
the chalcones from A. keiskei might be useful for
preventing the metabolic syndrome.
rial,9) anti-ulcer,10) anti-cancer11,12) and anti-inflamma-
tory13) actions. XA and 4HD have improved the
cholesterol metabolism of spontaneously hypertensive
rats.14,15) We have previously reported that XA and 4HD
induced adipogenesis in 3T3-L1 preadipocytes, and
enhanced the glucose uptake into mature adipocytes.16)
Mature adipocytes have recently been reported to
produce and secrete various hormones called ‘‘adipocy-
tokines.’’17) Adiponectin, one of the adipocytokines, has
enhanced glucose and lipid metabolism by binding its
receptors in liver and skeletal muscle.18,19) Since
adiponectin improves insulin resistance and dyslipide-
mia, the enhancing effect on adiponectin production can
be expected to prevent the metabolic syndrome. Recent
studies have shown that several phytochemicals en-
hanced adiponectin production in adipocytes.20–24) The
objective of this study was to isolate new chalcones
from A. keiskei and to evaluate their potential for
producing adiponectin in 3T3-L1 adipocytes.
Key words: Angelica keiskei; chalcone; adiponectin;
adipocyte
Chalcones are naturally occurring flavonoids with the
1,3-diphenyl prop-2-en-1-one structure. Chalcones are
synthesized from malonyl CoA and cinnamic acid by
chalcone synthase in higher plants, and are the starting
material for such flavonoids as flavanone, flavonol,
isoflavone, catechin and anthocyanidin. Recent studies
have revealed chalcones to have such bioactivities as
anti-bacterial, anti-fungal, anti-viral, anti-cancer, anti-
oxidative and anti-inflammatory effects.1–4) Chalcones
have very limited distribution in such edible plants as
Angelica keiskei Koidzumi (Ashitaba in Japanese),5)
hops,6) licorice7) and safflower.8)
The A. keiekei herb is grown along the Pacific coast of
Japan, and its leaves are used as a folk medicine and for
health-promoting foods. A. keiskei abundantly contains
such nutrients as vitamin A, vitamin K and dietary fiber,
as well as various chalcones, flavanones and coumarins
in its leaves, stems and roots. It has been reported that
the major chalcones, xanthoangelol (XA) and 4-hydro-
xyderricin (4HD), from A. keiskei exhibited anti-bacte-
Materials and Methods
Instruments and reagents. The 1H (600 MHz), 13C (150 MHz),
DEPT-135, DQF-COSY, TOCSY, HMQC and HMBC NMR spectra
were measured with an Avance 600 NMR spectrometer (Bruker,
Billerica, MA, USA). High-resolution MALDI-TOFMS data were
obtained with an Ultraflex II mass spectrometer (Bruker), using 2,5-
dihydroxybenzoic acid as a matrix. Dulbecco’s modified Eagle’s
medium (DMEM)-low glucose, ascorbic acid and dexamethasone were
purchased from Sigma-Aldrich (St. Louis, MO, USA). Calf serum
was purchased from Dainippon Sumitomo Pharma (Osaka, Japan),
and fetal bovine serum was purchased from Thermo Scientific
(Waltham, MA, USA). Triphenylphosphine was purchased from
Tokyo Chemical Industry (Tokyo, Japan), all other reagents being of
analytical grade.
Isolation of the chalcones from A. keiskei. A. keiskei was
cultivated in Kagoshima Prefecture in Japan. The dried root powder
of A. keisikei (20 kg) was extracted with 60 L of ethanol at room
temperature for 30 min. After filtration, the residue was extracted
twice with the same volume of ethanol. The ethanol extracts were
y
To whom correspondence should be addressed. Tel: +81-77-543-7201; Fax: +81-77-543-7238; E-mail: onogih@takara-bio.co.jp
Abbreviations: DEPT, distortionless enhancement by polarization transfer; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethylsulf-
oxide; DQF-COSY, double quantum filtered correlation spectroscopy; 4HD, 4-hydroxyderricin; HMBC, heteronuclear multiple bond correlation;
HMQC, heteronuclear multiple quantum correlation; MALDI-TOFMS, matrix assisted laser desorption ionization-time of flight mass spectrometry;
NMR, nuclear magnetic resonance; ODS, octadecyl silyl; PPAR, peroxisome proliferator-activated receptor; RT-PCR, reverse transcription
polymerase chain reaction; TOCSY, total correlated spectroscopy; XA, xanthoangelol

962
H. OHNOGI et al.
concentrated with a rotary evaporator. The resulting concentrate was
dissolved in 25% ethanol and subjected to ODS chromatography
(Cosmosil C-18 OPN, Nacalai Tesque, Kyoto, Japan), successively
eluting with 30, 40, 75 and 100% ethanol. The elute with 40%
ethanol from ODS chromatography was subjected to silica gel
chromatography (BW-300SP, Shin-Etsu Chemical, Tokyo, Japan)
and eluted with a gradient of chloroform/methanol (50:1–5:1, v/v)
to give 1, 21 mg; 2, 114 mg; 3, 27 mg; and 4, 70 mg. The elute
with 75% ethanol from ODS chromatography was subjected to silica
gel chromatography, eluting with chloroform/hexane (2:1 and 5:2)
and ethyl acetate to give 4HD, 8.7 g; 5, 22 mg; XA, 14.4 g; and 6,
245 mg.
Compound 5. Yellow amorphous powder; 1H-NMR (DMSO-d6) ꢀ:
1.40 (1H, m, H-500), 1.56 (1H, m, H-500), 1.62 (3H, s, 700-Me), 1.72 (3H,
s, 300-Me) , 1.89 (2H, m, H-400), 3.27 (1H, m, H-100), 3.31 (1H, m,
H-100), 3.91 (3H, s, 40-OMe), 4.07 (1H, t, J ¼ 6:9 Hz, H-600), 4.79 (1H,
s, H-800), 4.84 (1H, s, H-800), 5.14 (1H, t, J ¼ 6:6 Hz, H-200), 6.68 (1H,
d, J ¼ 9:0 Hz, H-50), 6.85 (2H, d, J ¼ 8:4 Hz, H-3, H-5), 7.78 (2H, d,
J ¼ 8:4 Hz, H-2, H-6), 7.78 (1H, d, J ¼ 15:0 Hz, H-ꢁ), 7.83 (1H, d,
J ¼ 15:0 Hz, H-ꢂ), 8.24 (1H, d, J ¼ 9:0 Hz, H-60), 10.15 (1H, s,
4-OH), 11.25 (1H, s, 600-OOH), 13.81 (1H, s, 20-OH). 13C-NMR
(DMSO-d6) ꢀ: 16.7 (300-Me), 17.7 (700-Me), 22.0 (C-100), 29.5 (C-500),
36.0 (C-400), 56.9 (40-OMe), 88.2 (C-600), 103.6 (C-50), 114.0 (C-800),
114.9 (C-10), 116.7 (C-30), 116.7 (2C, C-3, C-5), 118.1 (C-ꢂ), 122.9
(C-200), 126.5 (C-1), 131.3 (C-60), 132.3 (2C, C-2, C-6), 134.9 (C-300),
145.3 (C-700), 145.7 (C-ꢁ), 161.3 (C-4), 163.0 (C-20), 163.8 (C-40),
193.3 (C=O). HR MALDI-TOFMS m=z (M)þ: calcd. for C26H30O6:
438.2042; found: 438.2045.
Compound 6. Yellow amorphous powder; 1H-NMR (CDCl3) ꢀ:
1.34 (3H, s, 300-Me), 1.57 (2H, m, H-400), 1.65 (3H, s, 700-Me), 1.71 (3H,
s, 700-Me), 1.79 (1H, s, 300-OH), 2.11 (1H, m, H-500), 2.19 (1H, m,
H-500), 3.19 (2H, d, J ¼ 8:7 Hz, H-100), 4.82 (1H, t, J ¼ 8:7 Hz, H-200),
5.15 (1H, t, J ¼ 6:7 Hz, H-600), 5.21 (1H, s, 4-OH), 6.44 (1H, d,
J ¼ 8:4 Hz, H-50), 6.89 (2H, d, J ¼ 7:2 Hz, H-3, H-5), 7.46 (1H, d,
J ¼ 15:0 Hz, H-ꢂ), 7.58 (2H, d, J ¼ 7:2 Hz, H-2, H-6), 7.80 (1H, d,
J ¼ 8:4 Hz, H-60), 7.84 (1H, d, J ¼ 15:0 Hz, H-ꢁ), 13.51 (1H, s,
20-OH). 13C-NMR (CDCl3) ꢀ: 18.1 (700-Me), 22.4 (C-500), 23.2 (300-Me),
26.1 (700-Me), 27.3 (C-100), 37.1 (C-400), 74.2 (C-300), 91.6 (C-200), 102.1
(C-50), 114.2 (C-30), 115.4 (C-10), 116.4 (2C, C-3, C-5), 118.6 (C-ꢂ),
124.4 (C-600), 128.2 (C-1), 130.9 (2C, C-2, C-6), 132.1 (C-60), 132.7
(C-700), 144.3 (C-ꢁ), 158.3 (C-4), 161.9 (C-20), 167.0 (C-40), 192.5
(C=O). HR MALDI-TOFMS m=z (M)þ: calcd. for C25H28O5,
408.1937; found, 408.1928.
NMR and MS analyses of the chalcones. Compound 1. Yellow
amorphous powder; 1H-NMR (DMSO-d6) ꢀ: 1.18 (3H, s, 300-Me), 1.28
(3H, s, 300-Me), 3.07 (2H, m, H-100), 3.87 (3H, s, 40-OMe), 4.72 (1H, s,
300-OH), 4.78 (1H, t, J ¼ 8:7 Hz, H-200), 6.65 (1H, d, J ¼ 9:0 Hz, H-50),
6.82 (2H, d, J ¼ 8:4 Hz, H-3, H-5), 7.57 (2H, d, J ¼ 8:4 Hz, H-2, H-6),
7.59 (1H, d, J ¼ 15:6 Hz, H-ꢁ), 7.69 (1H, d, J ¼ 9:0 Hz, H-60), 7.81
(1H, d, J ¼ 15:6 Hz, H-ꢂ), 10.02 (1H, s, 4-OH). 13C-NMR (DMSO-d6)
ꢀ: 26.2 (300-Me), 26.8 (300-Me), 27.6 (C-100), 56.5 (40-OMe), 70.9
(C-300), 91.5 (C-200), 105.2 (C-50), 115.7 (C-30), 116.0 (C-10), 116.7 (2C,
C-3, C-5), 123.8 (C-ꢂ), 127.0 (C-1), 131.0 (2C, C-2, C-6), 131.3 (C-60),
142.7 (C-ꢁ), 160.5 (C-40), 160.6 (C-4), 161.8 (C-20), 186.5 (C=O). HR
MALDI-TOFMS m=z (M)þ: calcd. for C21H22O5, 354.1467; found,
354.1471.
Compound 2. Yellow amorphous powder; 1H-NMR (DMSO-d6) ꢀ:
1.20 (3H, s, 300-Me), 1.36 (3H, s, 700-Me), 1.57 (3H, s, 700-Me), 1.68
(2H, m, H-400), 2.10 (2H, m, H-500), 2.41 (1H, dd, J ¼ 9:0, 16.8 Hz,
H-100), 2.85 (1H, dd, J ¼ 6:0, 16.8 Hz, H-100), 3.76 (1H, m, H-200), 5.01
(1H, m, H-600), 5.23 (1H, d, J ¼ 4:8 Hz, 200-OH), 6.47 (1H, d,
J ¼ 8:4 Hz, H-50), 6.80 (2H, d, J ¼ 8:4 Hz, H-3, H-5), 7.38 (1H, d,
J ¼ 8:4 Hz, H-60), 7.44 (1H, d, J ¼ 15:6 Hz, H-ꢁ), 7.47 (1H, d,
J ¼ 15:6 Hz, H-ꢂ), 7.50 (2H, d, J ¼ 8:4 Hz, H-2, H-6), 9.96 (1H, s,
4-OH), 10.19 (1H, s, 40-OH). 13C-NMR (DMSO-d6) ꢀ: 18.1 (300-Me),
18.2 (700-Me), 22.1 (C-500), 26.3 (700-Me), 27.2 (C-100), 38.7 (C-400), 66.7
(C-200), 80.2 (C-300), 107.8 (C-50), 109.1 (C-30), 116.7 (2C, C-3, C-5),
121.0 (C-10), 125.1 (C-600), 125.1 (C-ꢂ), 127.0 (C-1), 130.3 (C-60),
130.8 (2C, C-2, C-6), 131.6 (C-700), 141.5 (C-ꢁ), 154.6 (C-20), 160.4
(C-4), 160.4 (C-40), 189.9 (C=O). HR MALDI-TOFMS m=z (M)þ:
calcd. for C25H28O5, 408.1937; found, 408.1934.
Compound 3. Yellow amorphous powder; 1H-NMR (DMSO-d6) ꢀ:
0.81 (3H, s, 700-Me), 1.03 (3H, s, 700-Me), 1.24 (3H, s, 300-Me), 1.54
(1H, m, H-500), 1.61 (1H, dd, J ¼ 4:8, 13.2 Hz, H-200), 1.71 (1H, m,
H-500), 1.75 (1H, m, H-400), 1.87 (1H, m, H-400), 2.34 (1H, dd, J ¼ 13:2,
16.8 Hz, H-100), 2.67 (1H, dd, J ¼ 4:8, 16.8 Hz, H-100), 3.27 (1H, m,
H-600), 4.65 (1H, d, J ¼ 4:8 Hz, 600-OH), 6.47 (1H, d, J ¼ 8:4 Hz,
H-50), 6.83 (2H, d, J ¼ 8:4 Hz, H-3, H-5), 7.39 (1H, d, J ¼ 8:4 Hz,
H-60), 7.42 (1H, d, J ¼ 15:6 Hz, H-ꢁ), 7.48 (1H, d, J ¼ 15:6 Hz, H-ꢂ),
7.51 (2H, d, J ¼ 8:4 Hz, H-2, H-6), 9.97 (1H, brs, 4-OH), 10.22 (1H,
brs, 40-OH). 13C-NMR (DMSO-d6) ꢀ: 15.3 (700-Me), 18.8 (C-100), 20.7
(300-Me), 28.1 (700-Me), 28.9 (C-500), 38.3 (C-400), 38.9 (C-700), 46.4
(C-200), 76.8 (C-600), 77.9 (C-300), 107.7 (C-50), 110.4 (C-30), 116.8 (2C,
C-3, C-5), 120.8 (C-10), 125.2 (C-ꢂ), 127.1 (C-1), 130.2 (C-60), 130.8
(2C, C-2, C-6), 141.2 (C-ꢁ), 154.9 (C-20), 160.3 (C-4), 160.6 (C-40),
189.8 (C=O). HR MALDI-TOFMS m=z (M)þ: calcd. for C25H28O5,
408.1937; found, 408.1932.
Reduction of 5. Compound 5 (1.8 mg) was treated by equivalent
triphenylphosphine in methanol at room temperature for 1 h. The
reaction product was subjected to TLC (Silica 60, Merck, Darmstadt,
Germany) in chloroform/methanol (100:1) to give compound 5R
(1.0 mg).
Cell culture. 3T3-L1 preadipocytes (JCRB 9014) were obtained
from Health Science Research Resources Bank (Tokyo, Japan) and
were maintained in DMEM-low glucose supplemented 10% calf
serum, 0.2 mM ascorbic acid, 50 U/mL of penicillin and 50 mg/mL of
streptomycin at 37 ꢀC in a humidified CO2 atmosphere. Two d after
confluence, the cells were differentiated for 5 d in DMEM-low
glucose containing 10% fetal bovine serum, 0.2 mM ascorbic acid and
0.25 mM dexamethasone (0–2 d) in the presence of chalcones dissolved
in DMSO or DMSO alone (0.1%). The medium was changed after
two d.
Adiponectin mRNA expression. Total RNA was isolated from
3T3-L1 cells by RNA Iso Plus (Takara Bio, Shiga, Japan). cDNA
was synthesized from total RNA by using the PrimeScript RT
reagent kit (Takara Bio, Shiga, Japan), and then subjected to real-
time RT-PCR using SYBR Premix Ex Taq II (Perfect Real Time;
Takara Bio) according to the manufacturer’s protocol with the Thermal
Cycler Dice real time system (Takara Bio). The PCR conditions were
as follows.
Compound 4. Yellow amorphous powder; 1H-NMR (DMSO-d6) ꢀ:
0.96 (3H, s, 700-Me), 1.02 (3H, s, 700-Me), 1.16 (1H, m, H-500), 1.61 (1H,
m, H-500), 1.73 (3H, s, 300-Me), 1.85 (1H, m, H-400), 2.15 (1H, m, H-400),
3.01 (1H, m, H-600), 3.24 (1H, m, H-100), 3.31 (1H, m, H-100), 4.00 (1H,
s, 700-OH), 4.23 (1H, d, J ¼ 6:0 Hz, 600-OH), 5.19 (1H, t, J ¼ 7:2 Hz,
H-200), 6.47 (1H, d, J ¼ 8:4 Hz, H-50), 6.84 (2H, d, J ¼ 8:4 Hz, H-3, H-
5), 7.75 (1H, d, J ¼ 5:4 Hz, H-ꢂ), 7.75 (1H, d, J ¼ 5:4 Hz, H-ꢁ), 7.75
(2H, d, J ¼ 8:4 Hz, H-2, H-6), 8.03 (1H, d, J ¼ 8:4 Hz, H-60), 10.11
(1H, s, 4-OH), 10.55 (1H, s, 40-OH), 14.00 (1H, s, 20-OH). 13C-NMR
(DMSO-d6) ꢀ: 17.0 (300-Me), 22.1 (C-100), 25.4 (700-Me), 27.2 (700-Me),
30.3 (C-500), 37.5 (C-400), 72.4 (C-700), 78.0 (C-600), 108.2 (C-50), 113.6
(C-10), 115.4 (C-30), 116.7 (2C, C-3, C-5), 118.3 (C-ꢂ), 122.4 (C-200),
126.7 (C-1), 130.7 (C-60), 132.0 (2C, C-2, C-6), 135.7 (C-300), 145.0
(C-ꢁ), 161.1 (C-4), 163.2 (C-40), 164.4 (C-20), 192.6 (C=O). HR
MALDI-TOFMS m=z (M)þ: calcd. for C25H30O6, 426.2042; found,
426.2036.
1 cycle of 95 ꢀC for 10 s, 45 cycles of 95 ꢀC for 5 s and 60 ꢀC for
30 s, followed by 1 cycle of 95 ꢀC for 15 s, 60 ꢀC for 30 s and 95 ꢀC for
15 s. ꢁ-Actin was used as an internal control gene. The primer
sequences were adiponectin (forward, 50-tgatggcagagatggcactc-30;
reverse, 50-cctgtcattccaacatctcc-30) and ꢁ-actin (forward, 50-ttctttgcagc-
tccttcgttg-30; reverse, 50-acatgccggagccgttg-30).
Adiponectin protein production. Adiponectin in the medium was
measured by using a commercial sandwich ELISA kit for mouse
adiponectin (R&D Systems, Minneapolis, MN, USA).
Statistical analysis. All data are expressed as the mean ꢁ SEM. The
statistical analysis was performed by using a one-way analysis of
variance (ANOVA) followed by Dunnett’s test. p < 0:05 was
considered as significant.

New Chalcones from Angelica keiskei
963
OH
OH
3
6
OH
2
4
5
HO
HO
5’
H3CO
4’
3’
1
6’
1’
β
1”
2”
1”
α
2’
2” O
1”
2”
3”
O
O
O
O
7”
HO
O
3”
4”
6”
3”
4”
HO
5”
5”
HO
6”
7”
2
1
3
OH
OH
R2
R1
O
2”
HO
3”
5”
1”
4”
OH
O
OH
O
6”
7”
R1
R2
6
OH
1”
1”
3”
5” 7”
OH
4
5
2”
2”
4”
6”
OH
3”
5”
6”
7”
OCH3
4”
8”
OOH
OH
XA
OCH3
4HD
Fig. 1. Structures of the Chalcones Isolated from A. keiskei.
Results and Discussion
three methyls, three methylenes, an aliphatic methine,
nine olefinic methines, an aliphatic quaternary, seven
olefinic quaternary carbons, and a carbonyl carbon. The
presence of a 20,30,4,40-tetrasubstituted chalcone skele-
ton was confirmed by the DQF-COSY and HMBC
Isolation and structural elucidation of the new
chalcones from A. keiskei
We isolated XA, 4HD and six new chalcones from the
ethanol extract of A. keiskei by ODS and silica gel
chromatography (Fig. 1). The structures of XA and 4HD
were confirmed by comparing with the literature data.25)
Compound 1 had the molecular formula of C21H22O5
determined by its HR MALDI-TOFMS value ([M]þ,
1
correlations as in the case of 1. The H- and 13C-NMR
spectra of 2 were very similar to those of xanthoangelol
I, 20, 30-[2-methyl-2-(4-methyl-3-pentenyl)-dihydropy-
rano]-4, 40-dihydroxychalcone, except for the presence a
hydroxyl group (ꢀH 5.23) at C-200 in 2.26) The functional
group linked to C-20 and C-30 was determined to be a
200,300,300-trisubsutituted dihydropyran ring as shown
Fig. 1 by DQF-COSY correlations (H-100/H-200; H-200/
200-OH; H-400/H-500; H-500/H-600) and HMBC long-range
correlations (H-100/C-20, C-30, C-40, C-200 and C-300;
200-OH/C-100, C-200 and C-300; 300-Me/C-200, C-300 and
C-400; H-400/C-500; H-600/700-Me; 700-Me/C-600, C-700 and
700-Me). The structure of 2 was therefore established to
be that shown in Fig. 1.
1
m=z 354.1471). The H-, 13C-NMR and HMQC spectra
indicated 1 to contain 21 carbons, including three
methyls, an aliphatic methylene, an aliphatic methine,
eight olefinic methines, an aliphatic quaternary, six
olefinic quaternary carbons, and a carbonyl carbon. The
DQF-COSY correlations (H-ꢂ/H-ꢁ; H-2/H-3; H-5/H-6;
H-50/H-60) and HMBC correlations (H-ꢂ/C-ꢁ, C=O
and C-1; H-ꢁ/C=O, C-1 and C-2, 6; H-2, 6/C-ꢂ, C-ꢁ,
C-1, C-2, 6, C-3, 5, and C-4; H-3, 5/C-1, C-2, 6 and C-3,
5; H-50/C-10, C-30 and C-40; H-60/C=O, C-20 and C-40)
revealed 1 to have a 20,30,4,40-tetra substituted chalcone
skeleton. The presence of 4-OH and 40-OMe were also
determined from the HMBC correlations (4-OH/C-3,
C-4 and C-5; 40-OMe/C-40). The functional group linked
to C-20 and C-30 was determined as a 200-(hydroxy
isopropyl)-dihydrofuran ring by the HMBC long-range
corr0e0 lations (H-100/C-20, C-30, C-40, C-200 and C-300;
H-2 /300-Me; 300-OH/C-200, C-300 and 300-Me; 300-Me/
C-200, C-300 and 300-Me). These results suggested the
structure of 1 to be that shown in Fig. 1.
Compound 3 had the molecular formula of C25H28O5
determined by its HR MALDI-TOFMS value ([M]þ,
1
m=z 408.1932). The H-, 13C-NMR, HMQC and DEPT
spectra indicated 3 to contain 25 carbons, including
three methyls, three methylenes, two aliphatic methines,
eight olefinic methines, two aliphatic quaternary, six
olefinic quaternary carbons, and a carbonyl carbon. The
presence of a trans-p-coumaroyl moiety in 3 was
confirmed by the DQF-COSY correlations (H-ꢂ/H-ꢁ;
H-2/H-3; H-5/H-6) and HMBC correlations (H-ꢂ/C-ꢁ,
C=O and C-1; H-ꢁ/C-ꢂ, C=O, C-1 and C-2, 6; H-2,
6/C-ꢁ, C-2, 6, C-3, 5 and C-4; H-3, 5/C-1, C-4 and C-3,
5). The presence of a hexahydroxanthene moiety in 3
was also indicated by the HMBC correlations (H-50/C-10
and C-30; H-60/C-20 and C-40; H-100/C-200 and C-300;
Compound 2 had the molecular formula of C25H28O5
determined by its HR MALDI-TOFMS value ([M]þ,
1
m=z 408.1934). The H-, 13C-NMR, HMQC and DEPT
spectra indicated 2 to contain 25 carbons, including

964
H. OHNOGI et al.
1
H-200/C-100, C-300 and C-700), DQF-COSY correlations
(H-50/H-60; H-100/H-100 and H-200; H-400/H-500; H-500/
H-600) and TOCSY correlation (H-400/H-600). 1H-, 13C
long-range couplings in the HMBC spectrum (300-Me/
C-200, C-300 and C-400; H-200/300-Me and 700-Me; H-600/
700-Me; 700-Me/C-200, C-600, C-700 and 700-Me) showed 3
to possess a methyl group at C-300 and gem-dimethyl
group at C-700 in the hexahydroxanthene moiety. More-
over, the HMBC correlations (600-OH/C-500, C-600 and
C-700), DQF-COSY correlations (600-OH/H-600) and
TOCSY correlations (600-OH/H-400 and H-500) indicated
the presence of a hydroxyl group at C-600. The structure
of 3 was therefore established to be that shown in Fig. 1.
Compound 4 had the molecular formula of C25H30O6
determined by its HR MALDI-TOFMS value ([M]þ,
(data not shown).27) The H- and 13C-NMR spectra of
5R were very similar to those of 5, except for the
presence of 600-OH (ꢀH 4.62) in 5R instead of 600-OOH
in 5 (ꢀH 11.25). These results elucidated 5 as 20,4-dihy-
droxy-30-(6-hydroperoxy-3,7-dimethyl-2,7-octadienyl)-40-
methoxychalcone.
Compound 6 had the molecular formula of C25H28O5
determined by its HR MALDI-TOFMS value ([M]þ,
m=z 408.1928). The H-, 13C-NMR and HMQC spectra
indicated 6 to contain 25 carbons, including three
methyls, three aliphatic methylenes, an aliphatic me-
thine, nine olefinic methines, an aliphatic quaternary,
seven olefinic quaternary carbons, and a carbonyl
carbon. The presence of a 20,30,4,40-tetrasubstituted
chalcone skeleton was confirmed by the DQF-COSY
and HMBC correlations, as in the case of 1. In addition,
20-OH and 4-OH were determined by their HMBC
correlations (20-OH/C-10, C-20 and C-30; 4-OH/C-3, C-4
and C-5). The functional group linked to C-30 and C-40
was determined to be a 200-substituted dihydrofuran ring
as shown Fig. 1 by the DQF-COSY correlations (H-100/
H-200; H-400/H-500; H-500/H-600) and HMBC long-range
correlations (H-100/C-20, C-30, C-40, C-200 and C-300;
300-Me/C-200, C-300 and C-400; H-400/C-300, C-500 and C-600;
H-500/C-400, C-600 and C-700; H-600/C-500 and 700-Me;
700-Me/C-600, C-700 and 700-Me). The structure of 6 was
thus established as that shown in Fig. 1.
1
1
m=z 426.2036). The H-, 13C-NMR and HMQC spectra
indicated 4 to contain 25 carbons, including three
methyls, three aliphatic methylenes, an aliphatic me-
thine, nine olefinic methines, an aliphatic quaternary,
seven olefinic quaternary carbons, and a carbonyl
carbon. The 1H- and 13C-NMR spectra of 4 were similar
to those of xanthoangelol, except for the signals arising
from the geranyl moiety. The HRMS data predicted that
4 corresponded to the dihydroxylated xanthoangelol.
13
The 1H-, C long-range couplings in the HMBC
spectrum (600-OH/C-500, C-600 and C-700; 700-OH/C-600,
C-700 and 700-Me) showed 4 to possess two hydroxyl
groups at C-600 and C-700. The HMBC correlations
(H-100/C-20, C-30, C-40, C-200 and C-300; H-200/C-100,
300-Me and C-400; 300-Me/C-200, C-300 and C-400; H-400/
C-200, C-300 and C-500; H-600/C-400; 700-Me/C-600, C-700 and
700-Me) and DQF-COSY correlations (H-100/H-200; H-400/
H-400 and H-500; H-500/H-500 and H-600) supported the
structure of the side chain shown Fig. 1. These results
elucidated 4 to be 20,4,40-trihydroxy-30-(6,7-dihydroxy-
3,7-dimethyl-2-octaenyl) chalcone.
This is the first report on the isolation of compounds
1–6 from a natural source, although the total chemical
synthesis of 3 has previously been reported.28)
Enhanced mRNA expression and protein secretion of
adiponectin by the chalcones from A. keiskei
The effects of XA, 4HD and the six new chalcones
isolated from A. keiskei were evaluated on adiponectin
production in 3T3-L1 adipocytes. Table 1 shows the
effects of the chalcones on adiponectin gene expression.
All the chalcones, except for compound 3, enhanced
the expression of adiponectin mRNA. In particular,
compound 4 (8.27-fold) and 6 (7.80-fold) markedly
increased the expression of adiponectin mRNA.
Figure 2 shows the protein concentration of adipo-
nectin protein in the supernatant of 3T3-L1 adipocytes
treated by the chalcones. Compounds 2, 3 and 6 showed
growth inhibition of 3T3-L1 adipocytes at 20 mM. All the
chalcones, except for compound 3, appreciably en-
hanced the production of adiponectin. In particular,
compounds 4 and 6 exhibited marked enhancement of
adiponectin production, compound 1 showing weak
Compound 5 had the molecular formula of C26H30O6
determined by its HR MALDI-TOFMS value ([M]þ,
1
m=z 438.2045). The H-, 13C-NMR and HMQC spectra
indicated 4 to contain 26 carbons, including three
methyls, three aliphatic methylenes, an olefinic methyl-
ene, an aliphatic methine, nine olefinic methines, eight
olefinic quaternary carbons, and a carbonyl carbon. The
1H- and 13C-NMR spectra of 5 were similar to those for
the xanthoangelol F, 30-geranyl-20,4-dihydroxy-40-me-
thoxychalcone structure, except for the signals arising
from the geranyl moiety.27) The structure of the side
chain was predicted to be the 6-hydroperoxy-3,7-
dimethyl-octa-2,7-di-ene moiety by HMBC correlations
(H-100/C-20, C-30, C-40, C-200 and C-300; H-200/300-Me and
C-400; 300-Me/C-200, C-300 and C-400; H-400/C-200, C-300 and
C-500; H-500/C-400; H-600/C-500, C-700 and C-800; 700-Me/
C-600 and C-700; H-800/C-600 and 700-Me) and DQF-COSY
correlations (H-100/H-200; H-400/H-500; H-500/H-500 and
H-600; H-800/700-Me). Baba et al. have also reported the
presence of chalcone with the hydroperoxylated side
chain, xanthoangelol E, in A. keiskei.25) To confirm the
presence of a hydroperoxy group at C-600, 5 was reduced
by triphenylphosphine, which converted the hydroper-
oxy group to a hydroxyl group, according to their
procedure. The reduced product (5R) was identified
as xanthoangelol G, 20,4-dihydroxy-30-(6-hydroxy-3,7-
dimethyl-2,7-octadienyl)-40-methoxychalcone by the
1H-, 13C- and HMBC NMR spectra and MS spectrum
Table 1. Effect of the Chalcones from A. keiskei on the Genetic
Expression of Adiponectin in 3T3-L1 Adipocytes
Compounds
Relative genetic expression
Control (DMSO)
1:00 ꢁ 0:08
1
2
3:43 ꢁ 0:45ꢂꢂ
5:15 ꢁ 0:31ꢂꢂꢂ
0:85 ꢁ 0:09
3
4
8:27 ꢁ 1:47ꢂꢂꢂ
3:92 ꢁ 0:49ꢂ
7:80 ꢁ 0:79ꢂꢂꢂ
5:27 ꢁ 0:20ꢂꢂꢂ
4:74 ꢁ 1:13ꢂꢂꢂ
5
6
XA
4HD
Data are shown as the mean ꢁ SEM (n ¼ 4{6).
ꢂ p < 0:05, ꢂꢂ p < 0:01, ꢂꢂꢂ p < 0:001 (vs. the Control)

New Chalcones from Angelica keiskei
965
60
50
40
30
20
10
0
***
***
***
***
***
***
***
***
10
***
***
***
5
***
***
***
5
**
**
*
*
5
10 20
1
5
10 20
4
5
10 20
5
5
10 20
XA
10 20
4HD
C
5
10
5
10
(µM)
2
3
6
Fig. 2. Effect of the Chalcones from A. keiskei on Adiponectin Production in 3T3-L1 Adipocytes.
3T3-preadipocytes were incubated for 5 d in DMEM containing 10% fetal bovine serum, 0.2 mM ascorbic acid and 0.25 mM dexamethasone in
the presence or absence of chalcones. Adiponectin in the medium was measured by ELISA. Data are shown as the mean ꢁ SEM (n ¼ 4{6),
ꢂ p < 0:05, ꢂꢂ p < 0:01, ꢂꢂꢂ p < 0:001 (vs. the Control).
activity and 3 having no activity. These results indicate
that the side chains of chalcones played an important role
in enhancing adiponectin production. We have also
previously indicated the structural importance of the side
chains to the activity of glucose uptake in adipocytes by
using various natural or synthetic chalcones.29)
Acknowledgment
We thank H. Hara for the MALDI-TOFMS measure-
ments and for valuable discussions.
References
The adipocyte-specific genetic expression including
adiponectin was regulated via the peroxisome prolifer-
ator-activated receptor (PPAR)-ꢃ signaling pathway.
Such thiazolidinediones as pioglitazone are powerful
PPAR-ꢃ agonists and enhance adipogenesis and adipo-
nectin production in 3T3-L1 adipocytes.30) Yamazaki
et al. have reported that synthetic phenolamides en-
hanced the expression of adiponectin mRNA in 3T3-L1
preadipocytes by activating the PPAR-ꢃ/retinoid X
receptor-ꢂ heterodimeric complex.31) However, we have
previously shown that XA and 4HD did not exhibit
PPAR-ꢃ activation in the GAL4-PPAR-ꢃ ligand domain
chimeric system.16) Moreover, our preliminary study
indicated that XA and 4HD enhanced the expression of
PPAR-ꢃ mRNA in 3T3-L1 preadipocytes, and that 4HD
and pioglitazone synergistically enhanced adiponectin
production (Ohnogi, H. and Tahara, K., unpublished
data). These results show that the chalcones from
A. keiskei may enhance adiponectin production by a
different mechanism from that of thiazolidinediones.
We have previously reported that orally administered
XA and 4HD suppressed the progression of diabetes in
KK-Ay type II diabetic mice.16) Moreover, an A. keiskei
extract containing chalcones improved insulin resistance
and tended to increase the serum adiponectin level in
fructose-drinking rats (Ohnogi, H. and Hayami, S.,
unpublished data). The results of our human study have
shown that 12 weeks ingestion of the green juice of
A. keiskei appreciably increased the serum adiponectin
level of mild diabetes when compared with placebo
juice of A. keiskei with the chalcones removed.32) Taken
together, it is possible that the chalcones from A. keiskei
prevented the metabolic syndrome as an adiponectin
inducer.
1) Batovska DI and Todorova IT, Curr. Clin. Pharmacol., 5, 1–29
(2010).
2) Dimmock JR, Elias DW, Beazely MA, and Kandepu NM, Curr.
Med. Chem., 6, 1125–1149 (1999).
3) Go ML, Wu X, and Liu XL, Curr. Med. Chem., 12, 483–499
(2005).
4) Kontogiorgis C, Mantzanidou M, and Hadjipavlou-Litina D,
Mini-Rev. Med. Chem., 8, 1224–1242 (2008).
5) Kozawa M, Morita N, Baba K, and Hata K, Yakugaku Zasshi
(in Japanese), 98, 210–214 (1978).
6) Stevens JF, Taylor AW, and Deinzer ML, J. Chromatogr. A,
832, 97–107 (1999).
7) Cuendet M, Guo J, Luo Y, Chen S, Oteham CP, Moon RC,
Breemen RB, Marler LE, and Pezzuto JM, Cancer Prev. Res., 3,
221–232 (2010).
8) Kazuma K, Takahashi T, Sato K, Takeuchi H, Matsumoto T,
and Okuno T, Biosci. Biotechnol. Biochem., 64, 1588–1599
(2000).
9) Inamori Y, Baba K, Tsujibo H, Taniguchi M, Nakata K, and
Kozawa M, Chem. Pharm. Bull., 39, 1604–1605 (1991).
10) Murakami S, Kijima H, Isobe Y, Muramatsu M, Aihara H,
Otomo S, Baba K, and Kozawa M, J. Pharm. Pharmacol., 42,
723–726 (1990).
11) Kimura Y and Baba K, Int. J. Cancer, 106, 429–437 (2003).
12) Kimura Y, Taniguchi M, and Baba K, Planta Med., 70, 211–219
(2004).
13) Shin JE, Choi EJ, Jin Q, Jin HG, and Woo ER, Arch.
Pharmacal. Res., 34, 437–442 (2011).
14) Ogawa H, Okada Y, Kamisako T, and Baba K, Clin. Exp.
Pharmacol. Physiol., 34, 238–243 (2007).
15) Ogawa H, Ohno M, and Baba K, Clin. Exp. Pharmacol.
Physiol., 32, 19–23 (2005).
16) Enoki T, Ohnogi H, Nagamine K, Kudo Y, Sugiyama K, Tanabe
M, Kobayashi E, Sagawa H, and Kato I, J. Agric. Food Chem.,
55, 6013–6017 (2007).
17) Matsuzawa Y, Funahashi T, Kihara S, and Shimomura I,
Arterioscler. Thromb. Vasc. Biol., 24, 29–33 (2004).
18) Kadowaki T and Yamauchi T, Endocr. Rev., 26, 439–451 (2005).
19) Kadowaki T, Yamauchi T, Kubota N, Hara K, Ueki K, and Tobe
K, J. Clin. Invest., 116, 1784–1792 (2006).
In conclusion, we isolated new chalcones from
A. keiskei and showed that they induced the production
of adiponectin in 3T3-L1 adipocytes. These chalcones
also displayed other functions of aldose reductase
inhibition and HMG-CoA reductase inhibition.33–35)
Further studies are required to clarify the beneficial
roles of the chalcones from A. keiskei in preventing the
metabolic syndrome and related diseases.
20) Miyata Y, Tanaka H, Shimada A, Sato T, Ito A, Yamanouchi T,
and Kosano H, Life Sci., 88, 613–618 (2011).
21) Hassan M, Yazidi CE, Landrier JF, Lairon D, Margotat A, and
Amiot MJ, Biochem. Biophys. Res. Commun., 361, 208–213
(2007).
22) Lee MJ, Rao YK, Chen K, Lee YC, and Tzeng YM,
J. Ethnopharmacol., 126, 79–85 (2009).

966
H. OHNOGI et al.
23) Ohara K, Uchida A, Nagasaka R, Ushio H, and Ohshima T,
Phytomedicine, 16, 130–137 (2009).
30) Ohsumi J, Sakakibara S, Yamaguchi J, Miyadai K, Yoshioka S,
Fujiwara T, Horikoshi H, and Serizawa N, Endocrinology, 135,
2279–2282 (1994).
24) Tzeng YM, Chen K, Rao YK, and Lee MJ, Eur. J. Pharmacol.,
607, 27–34 (2009).
31) Yamazaki Y, Kawano Y, and Uebayasi M, Life Sci., 82, 290–
300 (2008).
25) Baba K, Nakata K, Taniguchi M, Kido T, and Kozawa M,
Phytochemistry, 29, 3907–3910 (1990).
32) Ohnogi H, Enoki T, Hino F, and Kato I, Jpn. Pharmacol. Ther.
(in Japanese), 35, 647–660 (2007).
26) Akihisa T, Tokuda H, Hasegawa D, Ukiya M, Kimura Y,
Enjo F, Suzuki T, and Nishino H, J. Nat. Prod., 69, 38–42
(2006).
33) Ohnogi H, Sugiyama K, Muraki M, Enoki T, Sagawa H, and
Kato I, PCT Int. Appl. (in Japanese), WO2004/031165 (Apr.
15, 2004).
27) Nakata K, Taniguchi M, and Baba K, Nat. Med., 53, 329–332
(1999).
34) Ohnogi H, Sugiyama K, Enoki T, Kobayashi E, Sagawa H, and
Kato I, PCT Int. Appl. (in Japanese), WO2005/054170 (Jun. 16,
2005).
28) Topczewski JJ, Callahan MP, Neighbors JD, and Wiemer DF,
J. Am. Chem. Soc., 131, 14630–14631 (2009).
29) Enoki T, Ohnogi H, Kobayashi E, and Sagawa H, Nippon
Shokuhin Kagaku Kogaku Kaishi (in Japanese), 57, 456–463
(2010).
35) Enoki T, Kudo Y, Sugiyama K, Ohnogi H, Sagawa H, and Kato
I, PCT Int. Appl. (in Japanese), WO2005/074906 (Aug. 18,
2005).