Phenolics from Glycyrrhiza glabra roots and their PPAR-γ ligand-binding activity

Article abstract of DOI:10.1016/j.bmc.2009.11.027

Bioassay-guided fractionation of the EtOH extract of licorice (Glycyrrhiza glabra roots), using a GAL-4-PPAR-γ chimera assay method, resulted in the isolation of 39 phenolics, including 10 new compounds (1-10). The structures of the new compounds were determined by analysis of their spectroscopic data. Among the isolated compounds, 5′-formylglabridin (5), (2R,3R)-3,4′,7-trihydroxy-3′-prenylflavane (7), echinatin, (3R)-2′,3′,7-trihydroxy-4′-methoxyisoflavan, kanzonol X, kanzonol W, shinpterocarpin, licoflavanone A, glabrol, shinflavanone, gancaonin L, and glabrone all exhibited significant PPAR-γ ligand-binding activity. The activity of these compounds at a sample concentration of 10 μg/mL was three times more potent than that of 0.5 μM troglitazone.

Full text of DOI:10.1016/j.bmc.2009.11.027

Bioorganic & Medicinal Chemistry 18 (2010) 962–970
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Phenolics from Glycyrrhiza glabra roots and their PPAR-
c
ligand-binding activity
a,
b
b
b
Minpei Kuroda a, , Yoshihiro Mimaki , Shinichi Honda , Hozumi Tanaka , Shinichi Yokota ,
*
*
Tatsumasa Mae b
a School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1, Horinouchi, Hachioji, Tokyo 192 0392, Japan
b Frontier Biochemical & Medical Research Laboratories, Kaneka Corporation, 1-8 Miyamae-machi, Takasago-cho, Takasago, Hyogo 676 8688, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Bioassay-guided fractionation of the EtOH extract of licorice (Glycyrrhiza glabra roots), using a GAL-4-
Received 29 September 2009
Revised 10 November 2009
Accepted 10 November 2009
Available online 26 November 2009
PPAR-c chimera assay method, resulted in the isolation of 39 phenolics, including 10 new compounds
(1–10). The structures of the new compounds were determined by analysis of their spectroscopic data.
Among the isolated compounds, 50-formylglabridin (5), (2R,3R)-3,40,7-trihydroxy-30-prenylflavane (7),
echinatin, (3R)-20,30,7-trihydroxy-40-methoxyisoflavan, kanzonol X, kanzonol W, shinpterocarpin, licof-
lavanone A, glabrol, shinflavanone, gancaonin L, and glabrone all exhibited significant PPAR-
binding activity. The activity of these compounds at a sample concentration of 10 g/mL was three times
more potent than that of 0.5 M troglitazone.
c ligand-
Keywords:
Bioassay-guided fractionation
Phenolics
l
l
Ó 2009 Elsevier Ltd. All rights reserved.
Glycyrrhiza glabra
PPAR-c ligand-binding activity
1. Introduction
C57BL/6J mice, and spontaneously hypertensive rats, respectively.7
The activities were strongly suggested to be associated with PPAR-
Type 2 diabetes, obesity/abdominal obesity, hypertension, and
dyslipidemia are closely linked to insulin resistance. The clustering
of these risk factors in the same individual has been called meta-
bolic syndrome, which has been recognized as a major public
health problem around the world.1,2 A crucial role in development
of the metabolic syndrome is played by adipocytes, which are
highly specialized cells involved in energy regulation and homeo-
stasis. Adipocyte differentiation is a tightly controlled process in
which determinant genes such as those of peroxisome prolifera-
c
ligand-binding activity, and some prenylflavonoids including
glycycoumarin, glycyrin, dehydroglyasperin C, and dehydroglyas-
perin D were identified as PPAR-
ligands.7,8 Among them, glycy-
rin, one of the main PPAR- ligands of G. uralensis, significantly
c
c
decreased blood glucose levels in genetically diabetic KK-Ay mice.8
On the other hand, we found that a hydrophobic flavonoids-en-
riched fraction prepared from the EtOH extract of Glycyrrhiza glab-
ra L. roots, which exhibited PPAR-
c ligand-binding activity, was
also effective in preventing and/or ameliorating diabetes, abdomi-
nal obesity, and body weight gain in KK-Ay mice and/or high-fat
diet-induced obese C57BL/6J mice.9,10 These research findings
tor-activated receptor-
c (PPAR-c) and CCAAT/enhancer binding
protein-
a
lead to programmed adipocyte differentiation.3,4 PPARs
are ligand-dependent transcriptional regulatory factors belonging
to the nuclear receptor superfamily and regulate the expression
of a group of genes that maintain glucose and lipid metabolism.
prompted us to identify secondary metabolites with PPAR-c li-
gand-binding in the EtOH extract of G. glabra roots, which may
contribute to the anti-diabetes, anti-abdominal obesity, and anti-
obesity effects. Bioassay-guided fractionation of the EtOH extract
Among PPARs, PPAR-c is the predominant molecular target for
insulin-sensitizing thiazolidinedione drugs such as troglitazone,
pioglitazone, and rosiglitazone, which have been approved for
use in the treatment of type 2 diabetes patients.5,6 We previously
found that an EtOH extract of Glycyrrhiza uralensis F. roots, which
have long been used as a traditional Chinese medicine, was effec-
tive in preventing and/or ameliorating diabetes, abdominal obes-
ity, and hypertension in KK-Ay mice, high-fat diet-induced obese
using a GAL4-PPAR-c chimera assay method resulted in the isola-
tion of 10 new phenolic compounds (1–10), along with 29 known
ones, which were identified by comparison of their physical and
spectral data with those of reported compounds such as echinatin
(11),11 lichocalcone B (12),12 morachalcone A (13),13 20,3,40-trihy-
droxy-30- -dimethylallyl-600,600-dimethylpyrano[200,300:4,5]chalcone
c,c
(14),14 1-(20,40-dihydroxyphenyl)-2-hydroxy-3-(400-hydroxyphenyl)-
1-propanone (15),15 kanzonol Y (16),16 (3R)-vestitol (17),17 (3R)-
20,30,7-trihydroxy-40-methoxyisoflavan (18),18 kanzonol X (19),16
glabridin (20),19 40-O-methylglabridin (21),20 30-hydroxy-40-O-
methylglabridin (22),21 hispaglabridin A (23),20 hispaglabridin B
* Corresponding authors. Tel.: +81 426 76 4575; fax: +81 426 76 4579 (M.K.).
E-mail addresses: kurodam@toyaku.ac.jp (M. Kuroda), mimakiy@toyaku.ac.jp (Y.
Mimaki).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.027

M. Kuroda et al. / Bioorg. Med. Chem. 18 (2010) 962–970
963
(24),20 glabrene (25),22 kanzonol W (26),16 glabrocoumarin (27),23
R4
R
5'
5'
shinpterocarpin (28),24 O-methylshinpterocarpin (29),24 licoagro-
carpin (30),25 licoflavanone A (31),26 glabrol (32),19 shinflavanone
(33),24 euchrenone a5 (34),27 xambioona (35),28 gancaonin L (36),29
glabrone (37),30 kanzonol U (38),17 and 8,8-dimethyl-3,4-dihydro-
2H,8H-pyrano[2,3-f]-chromon-3-ol (39).31 This paper reports on
the structural determination of the 10 new compounds and the
4'
4' OH
OH
OH
6'
6'
R2
B
5
3 (5)
3'
3'
4
4
HO
HO
2 (6)
1
6
1
1'
1'
β
OH
β
2'
2'
A
5 (3)
3
α
R3
α
OH
1''
2''
1''
2''
6 (2)
2
4''
4''
OH
O
R1
O
3''
5''
3''
5''
R1 R2 R3
OH OMe
R4
H
R
3
4
PPAR-
c
ligand-binding activity of 1–39.
1
2
H
OH
prenyl'''
H
H
H
prenyl'''
2. Results and discussion
4''
5'
R2
5''
2''
4' OH
3''
6'
1''
B
8
8
3'
The dried roots of G. glabra (4.0 kg) were extracted with 95%
EtOH (20 L, 2ꢀ, 2 h) at 45 °C. Each extract was combined and con-
centrated under reduced pressure to give a 95% EtOH extract
7
9
HO
O
C
O
9
O
C
1''
2
1'
2
7
6
2'
A
A
6'
3
2''
4''
1'
2'
6
5' R1
10
10
3
OH
3''
5''
4
4
5
5
B
4'
O
HO
R1
CHO
H
OH
(120 g). The extract exhibited strong PPAR-
ity and its relative luminescence intensity was 2.2 at a sample con-
centration of 5 g/mL, which was almost as potent as that of
0.5 M troglitazone, a potent synthetic PPAR- agonist. The extract
c ligand-binding activ-
3'
R2
H
OH
7
5
6
l
l
c
4''
2''
was chromatographed on silica gel eluted with CHCl3–MeOH gra-
dients (19:1; 9:1; 2:1) and finally with MeOH. The CHCl3–MeOH
3''
1''
8
5''
OH
8
9
7
9
O
O
HO
O
C
2
7
(19:1) eluted portion (85 g) showed significant PPAR-
c ligand-
A
OH 2'
2'
1'
6'
6
1'
3'
4'
1''
10
3
binding activity (Fig. 1) and was subjected to a series of chromato-
graphic separations to obtain 1 (8.0 mg), 2 (13.8 mg), 3 (1.6 mg), 4
(6.9 mg), 5 (18.5 mg), 6 (8.6 mg), 7 (18.5 mg), 8 (4.9 mg), 9
(7.3 mg), 10 (30.2 mg), 11 (5.4 mg), 12 (17.3 mg), 13 (14.1 mg),
14 (17.4 mg), 15 (5.8 mg), 16 (2.5 mg), 17 (10.5 mg), 18 (8.1 mg),
19 (37.1 mg), 20 (193 mg), 21 (11.4 mg), 22 (54.7 mg), 23
(13.7 mg), 24 (10.7 mg), 25 (28.8 mg), 26 (3.0 mg), 27 (16.4 mg),
28 (41.0 mg), 29 (32.4 mg), 30 (6.1 mg), 31 (4.8 mg), 32
(13.5 mg), 33 (7.8 mg), 34 (1.5 mg), 35 (8.0 mg), 36 (8.8 mg), 37
(15.5 mg), 38 (21.6 mg), and 39 (17.4 mg) (Figs. 2 and 3).
Compound 1 was obtained as a yellow amorphous powder. Its
molecular formula was determined to be C21H22O6 on the basis
of its HRESIMS data, showing an accurate [M+H]+ ion at m/z
371.1487. The UV spectrum of 1 had absorption maxima at 366
and 248 nm, whereas the IR spectrum showed absorbance bands
at 3427 cmꢁ1 (hydroxy groups), 1625 cmꢁ1 (conjugated carbonyl
group), and 1595, 1507 and 1469 cmꢁ1 (aromatic rings). The 1H
NMR spectrum of 1 (acetone-d6) exhibited signals for two trans-
coupled protons at dH 7.91 and 7.67 (each d, J = 15.7 Hz), ortho-cou-
10
3
3'
4'
6
4
4
5
5
B
4''
2''
O
O
6'
5'
OMe
3''
5''
HO
OMe
5'
8
9
4'
HO
5'
2'
3'
1'
8
O
9
O
2
3
7
6
10
OH
10
Figure 2. Structures of 1–10.
pled aromatic protons at dH 7.25 and 6.72 (each d, J = 8.5 Hz), meta-
coupled aromatic protons at dH 7.55 and 7.51 (each d, J = 1.9 Hz),
and methoxy protons at dH 3.88 (s). In addition, the 1H NMR spec-
trum showed the presence of a prenyl (3-methyl-2-butenyl) group
[dH 5.40 (1H, m), 3.43 (2H, br d, J = 7.3 Hz), 1.77 and 1.75 (each 3H,
br s)]. These data suggest 1 to be a chalcone derivative with four
hydroxy groups, a methoxy group, and a prenyl group. In the
HMBC spectrum of 1 (Fig. 4), long-range correlations were ob-
served between H-2 (dH 7.51) and C@O (carbonyl, dC 188.0)/C-3
(dC 144.6)/C-4 (dC 148.3), and between H-6 (dH 7.55) and C@O/C-
1 (dC 130.7), indicating that two hydroxy groups are attached to
C-3 and C-4. HMBC correlations between H-100 (dH 3.43) and C-4/
C-6 (dC 122.8), between H-200 (dH 5.40) and C-5 (dC 128.2) are indic-
ative of the prenyl group at C-5. The structure of the ring-B moiety
assigned to 30,40-dihydroxy-20-methoxyphenyl and its linkage to C-
b of the trans-olefinic group were ascertained by long-range corre-
lations between H-50 (dH 6.72) and C-10 (dC 121.0)/C-30 (dC 138.7)/C-
40 (dC 149.0), between H-60 (dH 7.25) and C-b (dC 138.4)/C-20 (dC
148.7), and between methoxy protons and C-20. Thus, the structure
MeOH-Eluate Fr.
*
(2:1)-Eluate Fr.
*
(9:1)-Eluate Fr.
(19:1)-Eluate Fr.
EtOH Extr.
TRG
*
*
*
**
Cont.
of
1
was formulated as 3,30,4,40-tetrahydroxy-20-methoxy-5-
0
0.5
1
1.5
2
2.5
3
3.5
prenylchalcone.
Relative luminescent intensity
Compound 2, a yellow powder, was determined to be C25H28O5
on the basis of its HRESIMS data (m/z 409.2032 [M+H]+). The 1H
NMR spectrum of 2 showed two doublet signals at dH 7.75 and
7.64 (each d, J = 15.3 Hz), which are characteristic of the trans-ole-
finic group of chalcone derivatives. Furthermore, the 1H NMR spec-
trum exhibited signals for two prenyl groups at dH 5.38 and 5.29
(each 1H, m), 3.40 and 3.39 (each 2H, br d, J = 7.1 Hz), 1.79, 1.76,
1.74 and 1.66 (each 3H, br s), and a chelated hydroxy group at dH
12.31 (s), as well as signals for ortho-coupled aromatic protons at
Figure 1. PPAR-
ligand-binding activity of the licolice extract and fractions (5
that of troglitazone (TRG, 0.5 M) used as a positive control, were measured using a
GAL-4-PPAR- chimera assay. All samples were dissolved in dimethyl surfoxide,
and added to medium to obtain the indicated concentrations. The luminescence
intensity ratio (test group/control group) was determined for each sample, and
c
ligand-binding activity of the licorice extract and fractions. PPAR-
c
l
g/mL), as well as
l
c
PPAR-c ligand-binding activity was expressed as the relative luminescence
intensity of the test sample to that of the control sample. Values are means SD,
n = 4 experiments. Statistical significance is indicated as * (p <0.05) or ** (p <0.01) as
determined by Dunnett’s multiple comparison test.

964
M. Kuroda et al. / Bioorg. Med. Chem. 18 (2010) 962–970
R1
HO
O
OH
R4
O
O
O
OH
R2
R1
HO
R2
HO
HO
OH
OR3
HO
R3
HO
OR2
OH
R2
R1
O
R1
R2
H
Me
Me
H
OH
O
OH O
R1
H
H
R2
H
H
R3
Me
R4
H
Me OH
R1
H
H
R2
H
R3
OMe
OH OMe
R1
H
H
R1
R2
H
11
12
13 OH
17
18
20
21
14
H
15
16
prenyl
H
H
prenyl prenyl
19 prenyl prenyl OH
22 OH
prenyl
23
HO
O
O
HO
O
O
O
O
O
O
O
O
H
O
OH
OH
H
O
O
OH
HO
O
R
O
HO
OH
OH
R1
OH
OMe
24
25
26
27
28
29
OH
O
O
O
HO
O
HO
O
HO
O
HO
O
O
H
H
O
O
O
OMe
O
30
33
34
31
32
HO
O
HO
O
HO
O
O
O
OH
OH
O
O
OH
OH
O
OH
O
O
O
OH
O
35
36
37
38
39
Figure 3. Structures of 11–39.
dH 7.90 and 6.56 (each d, J = 8.9 Hz) and meta-coupled aromatic
protons at dH 7.20 and 7.19 (br s). The 13C NMR spectrum showed
a total of 25 carbon signals including a carbonyl carbon signal at dC
192.5. These data suggest that 2 is a diprenylated tetrahydroxy-
chalcone derivative. In the HMBC spectrum of 2 (Fig. 4), long-range
correlations were observed between H-100 (dH 3.39) and C-4 (dC
161.2), H-200 (dH 5.29) and C-3 (dC 113.9), H-6 (dH 7.90) and C-4/
C@O (dC 192.5), H-20 (dH 7.20) and C-30 (dC 145.1)/C-40 (dC 146.8)/
(1H ꢀ 2, d, J = 2.0 Hz) showed long-range correlations (Fig. 4). Thus,
the structure of 3 was characterized as 2,30,4,40,a-pentahydroxy-
3,50-diprenyl-dihydrochalcone.
Compound 4 exhibited a molecular formula of C20H22O6 on the
basis of its HRESIMS, which was less than that of 3 by C5H8, corre-
sponding to one prenyl group. The 1H NMR spectrum of 4 indicates
that it is a dehydrochalcone derivative closely related to 3. How-
ever, signals assignable to the prenyl group at C-50 of the ring B part
could not be detected in the 1H NMR spectrum of 4. Instead, signals
due to a 1,3,4-trisubstituted aromatic group were observed at dH
6.78 (1H, d, J = 2.0 Hz), 6.71 (1H, d, J = 8.1 Hz), and 6.57 (1H, d,
J = 8.1, 2.0 Hz), corresponding to H-20, H-50, and H-60, respectively.
The HMBC spectrum of 4 showed correlation peaks between H-20
C-
a
(dC 117.8), H-1000 (dH 3.40) and C-40 (dC 146.8)/C-60 (dC 123.4),
and between H-2000 (dH 5.38) and C-50 (dC 129.1), indicating that
two prenyl units are attached to C-3 and C-50, and four hydroxy
groups to C-2, C-4, C-30 and C-40, respectively. The structure of 2
was characterized as 2,30,4,40-tetrahydroxy-3,50-diprenylchalcone.
Compound 3 was obtained as a pale yellow powder and shown
to have a molecular formula of C25H30O6 on the basis of its HRE-
SIMS data (m/z 427.2133 [M+H]+). The 1H NMR spectrum of 3 dis-
played signals for ortho-coupled aromatic protons at dH 7.71 and
6.53 (each d, J = 8.9 Hz), meta-coupled aromatic protons at dH
6.64 and 6.49 (each d, J = 2.0 Hz), two prenyl groups at dH 5.27
(1H ꢀ 2, m), 3.37 and 3.26 (each 2H, br d, J = 7.2 Hz), and 1.78,
1.70, 1.69 and 1.65 (each 3H, br s), and a chelated hydroxy group
at dH 12.33 (s). Although these spectral features were similar to
and C-b (dC 41.9), H-60 and C-b, and between H-
a (dH 5.17) and
C-10 (dC 129.6) (Fig. 4). Thus, the structure of 4 was determined
to be 2,30,4,40,a-pentahydroxy-3-prenyl-dihydrochalcone.
Compounds 3 and 4 showed no specific rotations, thus being
obtained as racemic compounds.
Compound 5 was obtained as a yellow amorphous powder. Its
molecular formula was determined to be C21H20O5 on the basis
of its HRESIMS data. The 1H NMR spectrum of 5 exhibited charac-
teristic signals for an isoflavan skeleton at dH 4.43 (ddd, J = 10.2,
3.4, 2.2 Hz, H-2a), 4.07 (dd, J = 10.2, 10.2 Hz, H-2b), 3.54 (m, H-3),
3.07 (dd, J = 15.5, 11.1 Hz, H-4a) and 2.90 (ddd, J = 15.5, 5.0,
2.2 Hz, H-4b) as well as signals assignable to two aromatic protons
at dH 7.58 and 6.49 (each s), ortho-coupled aromatic protons at dH
6.87 and 6.32 (each d, J = 8.2 Hz) and a 2,2-dimethylpyran ring at
dH 6.63 and 5.65 (each 1H, d, J = 9.8 Hz), and dH 1.40 and 1.38 (each
those of 2, the trans-olefinic proton signals assignable to H-a and
H-b in 2 were displaced by three proton signals with an ABX-type
spin system at dH 5.16 (m), 2.99 (dd, J = 14.0, 4.8 Hz) and 2.82 (dd,
J = 14.0, 7.1 Hz) in 3. In the HMQC spectrum of 3, the proton signals
at dH 2.99 and 2.82 were associated with a methylene carbon signal
at d 42.1, with which the aromatic protons at d 6.64 and 6.49

M. Kuroda et al. / Bioorg. Med. Chem. 18 (2010) 962–970
965
H
H
H
H
H
H
OH
OH
H
H
OH
OH
H
H
HO
HO
OH
H
H
OMe
H
H
H
H
H
H
O
OH
O
H
H
1
2
H
H
H
H
H
OH
H
OH
H
H
H
H
H
HO
HO
OH
OH
H
H
H
H
H
H
H
H
OH
OH
OH
O
OH
O
H
H
3
4
H
H
H
H
OH
H
H
H
O
H
O
H
H
H
HO
H
O
O
H
H
H
H
H
C
O
H
OH
H
H
H
H
H
HO
OH
5
7
H
H
OH
HO
O
H
H
O
H
O
O
H
H
H
OH
H
H
O
H
OMe
H
HO
OMe
H
9
8
Figure 4. Key HMBC correlations of 1–5 and 7–9.
3H, s). These 1H NMR spectral features of 5 are very similar to those
of glabridin (20). In addition, the presence of a formyl group in the
molecule of 5 was shown by the 1H and 13C NMR signals at dH 9.77
and dC 195.3, and it was attached to the C-50 position as deter-
mined by HMBC correlations between the formyl proton signal
and C-40 (dC 163.6)/C-50 (dC 115.4)/C-60 (dC 133.9) (Fig. 4). Since
the CD profile of 5 was the same as that of synthetic 50-formylgla-
bridin prepared from 20 by formylation, the absolute configuration
at C-3 was determined to be R. Thus, the structure of 5 was as-
signed as 50-formyl glabridin.
that of 20, one of the germinal dimethyl groups linked to C-300 at dH
1.39 and 1.38 disappeared in 6, and resonances for a hydroxy-
methyl group were detected at dH 3.63 and 3.54 (each d,
J = 11.3 Hz). These 1H NMR spectroscopic data, along with a
hydroxymethyl carbon signal at dC 67.7 and HMBC correlations be-
tween H2–400 (dH 3.63 and 3.54) and C-200 (dC 126.1)/C-300 (dC 78.8)/
C-500 (dC 22.5), allowed the structure of 6 to be assigned as 400-
hydroxyglabridin. The CD spectrum of 6 was consistent with that
of 20, giving evidence of the 3R configuration.
Compound 7 was isolated as a yellow powder and its molecular
formula was determined to be C20H20O5 by its HRESIMS. The UV
spectrum of 7 had absorption maxima at 313 and 276 nm, whereas
the IR spectrum showed absorption bands at 3374 cmꢁ1 (hydroxy
Compound 6 had a molecular formula of C20H20O5 on the basis
of its HRESIMS, which differed from 20 by containing one more
oxygen atom. When the 1H NMR spectrum of 6 was compared with

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M. Kuroda et al. / Bioorg. Med. Chem. 18 (2010) 962–970
groups), 1673 cmꢁ1 (a carbonyl group), and 1608, 1502 and
1463 cmꢁ1 (aromatic rings). The 1H NMR spectrum of 7 showed
signals assignable to a prenyl group at dH 5.39 (1H, m), 3.38 (2H,
d, J = 7.3 Hz), and 1.74 and 1.72 (each 3H, br s), and two methines
bearing an oxygen function at dH 5.03 and 4.59 (each d,
J = 11.9 Hz)]. Furthermore, six aromatic protons comprising two
ABX-type spin-coupling systems at dH 7.74 (d, J = 8.6 Hz), 6.64
(dd, J = 8.6, 2.2 Hz), and 6.41 (d, J = 2.2 Hz); dH 7.35 (d, J = 2.0 Hz),
7.27 (dd, J = 8.2, 2.0 Hz), and 6.90 (d, J = 8.2 Hz), were assigned to
two 1,3,4-trisubstituted aromatic rings. From the above data, 7
was deduced to be dihydroxyflavan-3-ol derivative with a prenyl
unit. HMBC correlations between H-5 (dH 7.74) and C-4 (dC
192.7)/C-9 (dC 164.1), H-8 (dH 6.41) and C-7 (dC 165.4)/C-9, H-6
(dH 6.64) and C-7, H-20 (dH 7.35) and C-2 (dC 84.6)/C-40 (dC 155.8),
H-60 (dH 7.27) and C-2/C-40, and between H-100 (dH 3.38) and C-20
(dC 130.0)/C-30 dC 128.1)/C-40 indicate that two hydroxy groups
and a prenyl group are attached to C-7, C-40, and C-30, respectively
(Fig. 4). The positive Cotton effects at 210, 240, and 334 nm and
negative Cotton effect at 304 nm in the CD spectrum of 7 allowed
the absolute configurations to be assigned as 2R and 3R.32 Thus, the
structure of 7 was determined to be (2R,3R)-3,40,7-trihydroxy-30-
prenylflavanone.
Compound 8 (C21H20O5) was suggested to be an isoflavone
derivative by the UV absorption maximum at kmax 263 nm,33 and
a proton resonance at dH 8.16 (1H, s) and its corresponding
oxygen-bearing olefinic carbon signal at dC 152.2. The 1H NMR
spectrum contained signals for an aromatic proton at dH 7.54 (s),
p-disubstituted aromatic protons at dH 7.55 and 6.98 (each d,
J = 8.8 Hz) and methoxy protons at d 3.84 (3H, s). In addition, the
1H NMR spectrum implied the presence of a prenyl unit [dH 5.41
(1H, m), 3.56 2Y, d, J = 7.3 Hz), and 1.76 (3H x 2, br s)]. In the HMBC
spectrum of 8 (Fig. 4), correlation peaks were observed between
H-5 (dH 7.54) and C-4 (dC 175.4)/C-7 (dC 148.7)/C-9 (dC 145.7)/
C-100 (dC 28.4), between methoxy protons (dH 3.84) and C-40
(dC 159.9), and between H-2 (dH 8.16) and C-8 (dC 145.7), indicating
two hydroxy groups to be located at C-7 and C-8, a methoxy
group at C-40, and a prenyl group at C-6, respectively. Thus, the
structure of 8 was determined to be 7,8-dihydroxy-40-methoxy-
6-prenylisoflavanone.
30), ortho-coupled protons at dH 7.71 and 6.52 (each d, J = 8.7 Hz),
a 2,2-dimethylpyran ring at dH 6.65 and 5.76 (each 1H, d,
J = 10.1 Hz), and dH 1.46 and 1.45 (each 3H, s), and a methoxy
group at dH 3.74 (3H, s). The 1H and 13C NMR spectral data of 9
were essentially analogous to those of glabroisoflavanone B;21
however, slight differences were recognized in the signals due to
the ring C part between the two compounds. Furthermore, the
molecular formula of 9 was higher than that of glabroisoflavanone
B by one oxygen atom and an oxygenated quaternary carbon signal
was observed at dC 74.5. A pair of oxymethylene proton signals at
dH 4.99 and 4.41 (each d, J = 11.8 Hz) were associated with the cor-
responding oxymethylene carbon signal at dC 74.4 (C-2) by the
HMQC spectrum, and a long-range correlation was observed from
H-60 (dH 7.39) to C-3 (dC 74.5) in the HMBC spectrum. Thus, the
presence of a hydroxy group at C-3 was verified, and the structure
of 9 was established as 20,3-dihydroxy-40-methoxy-300,300-dimethyl-
pyrano[200,300:7,8]isoflavanone. Compound 9 showed no specific
rotation nor Cotton effects in the CD spectrum, which is indicative
of a racemate.
Compound 10 (C14H16O4) was suggested to be a chromone
derivative whose structure was closely related to known com-
pound 39 on the basis of its spectroscopic data. However, the
molecular formula of 10 was higher than that of 39 by one oxygen
atom. On comparison of the 1H NMR spectrum of 10 with that of
39, one of the germinal dimethyl group attached to C-30 at d 1.23
and 1.22 (each s) could not be observed in 10, and signals for a
hydroxymethyl group appeared at d 3.62 and 3.54 (each d,
J = 11.3 Hz). HMBC correlations between H2-40 (dH 3.62 and 3.54)
and C-20 (dC 126.2)/C-30 (dC 78.8)/C-50 (dC 22.5) allowed the struc-
ture of 10 to be assigned as 8-hydroxymethyl-8-methyl-3,4-dihy-
dro-2H,8H-pyrano[2,3-f]-chromon-3-ol. Compound 10 showed no
specific rotation and was obtained as a racemate.
3. PPAR-c ligand-binding activity
Compounds 5, 7, 11, 18, 19, 26, 28, 31–33, 36, and 37 exhibited
significant PPAR- ligand-binding activity, among which the pre-
c
nylflavone derivative, licoflavanone A (31), showed the most po-
tent ligand-binding activity (Fig. 5). The isoflavone derivative,
kanzonol X (19), and flavanone derivative, glabrol (32), both having
two prenyl units, also showed potent ligand-binding activity. The
activity of hispaglabridin B (24) and xambioona (35), in which
the two prenyl units are cyclized to form two six-membered rings,
Compound 9 had a molecular formula of C21H20O6, as deter-
mined by its HRESIMS. The 1H NMR displayed signals for a 1,2,4-
trisubstituted aromatic ring at dH 7.39 (1H, d, J = 8.6 Hz, H-60),
6.44 (1H, dd, J = 8.6, 2.4 Hz, H-50) and 6.41 (1H, d, J = 2.4 Hz, H-
16
14
**
**
**
**
12
**
**
**
10
**
**
** **
**
8
**
**
**
**
6
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
4
2
0
**
**
**
**
**
**
**
**
**
*
**
**
**
*
**
**
*
**
*
*
**
*
*
*
**
*
Figure 5. PPAR-
c
ligand-binding activity of compounds 1–39 at 2 ( ) and 10 (
)
l
g/mL with the GAL-4-PPAR-
c
chimera assay. Troglitazone at 0.5, 1.0, and 2.0
lM was used
as a positive control, and dimethyl sulfoxide at 1 mL/L as a solvent control. Values are means SD, n = 4 experiments. Statistical significance is indicated as * (p <0.05) or ** (p
<0.01) as determined by Dunnett’s multiple comparison test.

M. Kuroda et al. / Bioorg. Med. Chem. 18 (2010) 962–970
967
were less than that of 19 and 32, implying that the two prenyl
units are necessary for the appearance of the potent activity in
these compounds. As we have previously mentioned, the isoprenyl
group at C-6 and the hydroxy group at C-20 in the aromatic ring-C
part in the isoflavan, isoflavene, or arylcoumarin skeleton have
nosy at Tokyo University of Pharmacy and Life Sciences. A voucher
specimen (No. 02-5-GG) is maintained at the School of Pharmacy,
Tokyo University of Pharmacy and Life Sciences.
5.3. Extraction and isolation
been found to be the structural requirements for PPAR-
binding activity in the phenolics isolated from G. uralensis roots.8
Considering all of the above data together, PPAR- ligand-binding
activity in the phenolic compounds are affected by the slight differ-
ences of substitution groups on the aromatic rings.
c ligand-
The dried roots of G. glabra (4.0 kg) were extracted with EtOH
(20 L, 45 °C, 2 h). The EtOH extract (120 g) was chromatographed
on silica gel eluted with CHCl3–MeOH gradients (19:1; 9:1; 2:1),
and finally with MeOH alone, to give four fractions (I–IV). Since
the activity was found to be concentrated in fraction I (85 g), the
fraction was subjected to silica gel column chromatography
eluted with CHCl3–MeOH (99:1), and finally with MeOH alone,
to give eight fractions (I-A–H). Fraction I-B (12 g) was separated
by silica gel column chromatography using hexane–Me2CO (3:1)
to afford eight fractions (I-B1–8). Fraction I-B2 (1.3 g) was sub-
jected to silica gel column chromatography eluted with hex-
ane–EtOAc (10:1) to give seven fractions (I-B2a–g). Fraction I-
B2c (44.7 mg) was subjected to preparative HPLC (flow rate,
0.8 mL/min) using MeOH–H2O (20:1) to give 29 (32.4 mg, tR
34 min). Fraction I-B2e (274 mg) was subjected to preparative
HPLC using MeOH–H2O (4:1) to give 35 (8.0 mg, tR 126 min).
Fraction I-B4 (3.5 g) was separated by ODS silica gel column
chromatography eluted with MeCN–H2O (2:1) to give five frac-
tions (I-B4a–e). Fraction I-B4a (1.02 g) was subjected to ODS sil-
ica gel column chromatography eluted with MeCN–H2O (2:1) to
give nine fractions (I-B4a1–9). Compound 39 (17.4 mg, tR
29 min) was obtained from fraction I-B4a1 (92.8 mg) by subject-
ing it to preparative HPLC using MeCN–H2O (2:1). Fraction I-
B4a4 (83.7 mg) was subjected to preparative HPLC using
MeCN–H2O (2:1) to give 28 (41.0 mg, tR 45 min) and 30
(6.1 mg, tR 58 min). Similarly, purification of fraction I-B4a5
c
4. Conclusion
The EtOH extract of G. glabra roots exhibited considerable PPAR-
c
ligand-binding activity and bioassay-guided fractionation of the
extract resulted in the isolation of nine chalcones (1–4, 11–16),
10 isoflavans (5, 6, 17–24), an isoflaven (25), two 3-arylcoumarin
(26, 27), three pterocarpans (28–30), a flavone (31), a flavanol
(7), four flavanones (32–35), three isoflavone (8, 36, 37), an isoflav-
ane (9), an 2-arybenzofuran (38), and two chromones (10, 39),
including 10 new compounds (1–10). It is notable that 5 is the first
naturally occurring isoflavan with a formyl group at the B ring part.
Compounds 13, 15, 17, 18 and 36 were isolated from G. glabra for
the first time. The isolated compounds were evaluated for their
PPAR-
c
lignd-binding activity. Compounds 5, 7, 11, 18, 19, 26, 28,
ligand-binding activ-
31–33, 36, and 37 showed significant PPAR-
c
ity, among which the prenylflavone derivative, licoflavone A (31),
showed the most potent ligand-binding activity. These active com-
pounds were considered to contribute mainly to the PPAR-
c li-
gand-binding activity of the EtOH extract, which suggested that
the hydrophobic phenolics of G. glabra roots showed anti-diabetes,
anti-abdominal obesity and anti-obesity effects in disease animal
(124 mg) yielded
6 (8.6 mg, tR 53 min), and fraction I-B4c
models, possibly mediated via activation of PPAR-c.
(70 mg) gave 21 (11.4 mg, tR 67 min), 23 (13.7 mg, tR 81 min),
24 (10.7 mg, tR 133 min) and 33 (7.8 mg, tR 87 min). Fraction I-
B6 (1.8 g) was separated by ODS silica gel column chromatogra-
phy eluted with MeCN–H2O (3:1) to give eight fractions (I-B6a–
h). Fraction I-B6b (215 mg) was subjected to preparative HPLC
5. Experimental
5.1. General experimental procedures
using MeCN–H2O (2:1) to give
9 (7.3 mg, tR 33 min), 19
Optical rotations were measured using a JASCO P-1030 (Tokyo,
Japan) automatic digital polarimeter. IR spectra were recorded on a
JASCO FT-IR 620 spectrophotometer, UV spectra on a JASCO V-630
spectrophotometer, and CD spectra on a JASCO J-720 instrument.
NMR spectra were recorded on a Bruker DRX-500 spectrometer
(500 MHz for 1H NMR, Karlsruhe, Germany) using standard Bruker
pulse programs. Chemical shifts are given as d-value with reference
to tetramethylsilane (TMS) as an internal standard. HRESIMS data
were obtained on a Waters-Micromass LCT mass spectrometer
(Manchester, UK). Diaion HP-20 (Mitsubishi-Chemical, Tokyo,
Japan), silica gel (Fuji-Silysia Chemical, Aichi, Japan), and ODS silica
gel (Nacalai-Tesque, Kyoto, Japan) were used for column chroma-
tography (CC). TLC was carried out on precoated Silica Gel 60
F254 (0.25 mm, Merck, Darmstadt, Germany) and RP-18 F254S
(0.25 mm thick, Merck) plates, and spots were visualized by spray-
ing with 10% H2SO4 followed by heating. HPLC was performed by
using a system comprised of a CCPM pump (Tosoh, Tokyo, Japan),
a CCP PX-8010 controller (Tosoh), an RI-8010 detector (Tosoh),
and a Rheodyne injection port. A Capcell Pak C18 UG120 column
(37.1 mg, tR 51 min) and 22 (54.7 mg, tR 58 min). Similarly, puri-
fication of fraction I-B6d (201 mg) yielded 14 (17.4 mg, tR
83 min) and 34 (1.5 mg, tR 79 min). Fraction I-C (13.2 g) was
subjected to silica gel column chromatography eluted with
CHCl3–MeOH (99:1), and finally with MeOH alone, to give seven
fractions (I-C1–7). Fraction I-C5 (1.5 g) was subjected to ODS sil-
ica gel column chromatography eluted with MeOH–H2O (5:1)
and MeCN–H2O (1:1) to give three fractions (I-C5a–c). Fraction
I-C5a (458 mg) was chromatographed on ODS silica gel eluted
with MeOH–H2O (5:2) to give a mixture of 8, 16, and 37, which
was then separated by preparative HPLC using MeOH–H2O (5:2)
to yield 8 (4.9 mg, tR 72 min), 16 (2.5 mg, tR 119 min) and 37
(15.5 mg, tR 82 min). Compound 32 (13.5 mg) was obtained from
fraction I-C5c (350 mg) by subjecting it to ODS silica gel column
chromatography eluted with MeOH–H2O (5:1). Fraction I-D
(11.3 g) was subjected to an ODS silica gel column eluted with
MeOH–H2O (4:1; 7:3) and MeCN–H2O (1:1), a silica gel column
with hexane–Me2CO (2:1), and to preparative HPLC using
MeCN–H2O (1:1) to afford 13 (14.1 mg). Fraction I–E (10.7 g)
was subjected to ODS silica gel column chromatography using
MeCN–H2O (3:2) to afford four fractions (I-E1–4). Fraction I-E1
(756 mg) was separated by ODS silica gel column chromatogra-
phy eluted with MeCN–H2O (2:3) to give seven fractions (I-
E1a–g). Purification of fractions I-E1a (77.7 mg), I-E1c (55.4 mg)
and I-E1d (171 mg) by preparative HPLC using MeOH–H2O
(7:3) led to the isolation of compounds 10 (30.2 mg, tR
22 min), 18 (8.1 mg, tR 32 min), and 7 (18.5 mg, tR 48 min) and
(10 mm i.d. ꢀ 250 mm, 5
lm, Shiseido, Tokyo, Japan) was used
for preparative HPLC.
5.2. Plant material
The roots of G. glabra were collected in the northwestern re-
gions of the People’s Republic of China in May 2002 and identified
by Dr. Yutaka Sashida, emeritus professor of Medicinal Pharmacog-

Table 1
1H NMR spectral data of 1–10 in acetone-d6
Position
1
2
3
4
Position
2
5
6
7
8
9
10
J (Hz)
dH
J (Hz) dH
J (Hz) dH
J (Hz)
dH
J (Hz)
dH
J (Hz)
dH
J (Hz)
dH
J (Hz) dH
J (Hz) dH
J (Hz) dH
1
2
3
4
5
6
—
7.51 d
—
—
—
7.55 d
—
—
—
—
—
—
—
—
—
4.43 ddd
4.07 dd
3.54 m
3.07 dd
2.90 ddd
6.87 d
6.32 d
—
—
—
—
—
10.2, 3.4, 2.2 4.34 ddd 10.2, 3.3, 2.1 5.03 d
10.2, 10.2
11.9
11.9
8.16 s
—
—
—
4.99 d
11.8
11.8
4.16 m
3.90 m
4.13 m
1.9
1.9
—
—
—
—
—
—
4.03 dd 10.2, 10.2
3.47 m
2.99 dd 15.1, 11.0
4.41 d
—
—
3
4
4.59 d
—
15.5, 11.1
15.5, 5.0, 2.2 2.80 ddd 15.1, 4.9, 1.9
8.2
8.2
—
—
2.95 dd 15.7, 4.6
2.63 dd 15.7, 6.7
6.78 d 8.2
6.28 d 8.2
6.56 d
7.90 d
12.31 s
—
8.9
8.9
6.53 d
8.9
8.9
6.55 d
7.76 d
12.33 s
—
6.78 d
—
8.9
8.9
7.71 d
12.33 s
—
5
6
7
8
6.84 d
6.29 d
—
—
—
—
—
—
8.2
8.2
7.74 d
6.64 dd
—
6.41 d
—
—
—
7.35 d
—
—
8.6
8.6, 2.2
7.54 s
—
—
7.71 d
8.7
8.7
2-OH
10
20
30
40
50
60
a
6.52 d
—
—
—
—
—
—
—
—
—
—
7.20 br s
6.64 d
—
—
—
6.49 d
2.0
2.0
2.2
2.0
8.2
—
—
—
—
7.55 d
6.98 d
—
—
6.98 d
—
—
9
—
—
10
6.72 d
7.25 d
7.67 d
7.91 d
8.5
8.5
—
6.71 d
6.57 dd
5.17 m
8.1
10
6.68 d 10.1
5.64 d 10.1
—
3.62 d 11.3
3.54 d 11.3
7.19 br s
2.0
8.1, 2.0 20
30
—
6.49 s
—
8.8
8.8
—
6.41 d
—
15.7 7.64 d
15.7 7.75 d
15.3 5.16 m
15.3 2.99 dd
2.82 dd
6.47 d
—
2.3
2.4
b
14.0, 4.8 3.02 dd
14.0, 7.1 2.81 dd
—
14.0, 4.4 40
14.0, 7.7
50
C@O
100
—
—
—
—
6.36 dd 8.3, 2.3
6.90 d
7.27 dd
8.8
8.8
6.44 dd
7.39 d
6.65 d
5.76 d
—
8.6, 2.4
8.6
3.43 d (2H) 7.3
5.40 m
3.39 d (2H) 7.1
5.29 m
3.37 d (2H) 7.2
5.27 m
3.37 d (2H) 7.2
5.27 m
60
7.58 s
6.63 d
5.65 d
—
6.98 d
6.71 d
5.64 d
—
3.63 d
3.54 d
1.36 s
8.3
10.0
10.0
8.2, 2.2 7.55 d
1.34 s
200
100
200
300
400
9.8
9.8
3.38 d (2H) 7.3
5.39 m
3.56 d (2H) 7.3
5.41 m
10.1
10.1
300
—
—
—
—
400
1.77 br s
1.75 br s
1.66 br s
1.79 br s
3.40 d (2H) 7.1
5.38 m
1.65 br s
1.78 br s
3.26 d (2H) 7.2
5.27 m
1.66 br s
1.78 br s
—
—
500
1.40 s
11.3
11.3
—
1.72 br s
1.76 br s
1.46 s
1000
2000
3000
4000
5000
OMe
500
CHO
OMe
1.38 s
9.77 s (1H)
1.74 br s
1.76 br s
1.45 s
—
—
1.76 br s
1.74 br s
1.70 br s
1.69 br s
3.84 s (3H)
3.74 s (3H)
3.88 s (3H)

M. Kuroda et al. / Bioorg. Med. Chem. 18 (2010) 962–970
969
17 (10.5 mg, tR 42 min), respectively. Fraction I-E1f (84.7 mg)
was subjected to preparative HPLC (flow rate, 0.6 mL/min) with
MeOH–H2O (8:2) to give 31 (4.8 mg, tR 50 min). Fraction I-E2
(2.9 g) was separated by silica gel column chromatography
eluted with CHCl3–EtOAc (10:1) to give nine fractions (I-E2a–i).
Purification of fraction I-E2b (160 mg), I-E2f (261 mg) and I-
E2 g (305 mg) by preparative HPLC (flow rate, 0.7 mL/min) with
MeCN–H2O (3:2) to give 25 (28.8 mg, tR 39 min) and 38
(21.6 mg, tR 44 min), 26 (3.0 mg, tR 42 min) and 3 (1.6 mg, tR
67 min), respectively. Fraction I-E3 (6.5 g) was subjected to a
Sephadex LH-20 column eluted with MeOH and an ODS silica
gel column with MeCN–H2O (3:2), and to preparative HPLC using
MeCN–H2O (2:1) to give 27 (16.4 mg, tR 38 min). Fraction I-E4
(1.6 g) was subjected to an ODS silica gel column eluted with
MeCN–H2O (2:1) and a silica gel column with CHCl3–MeOH
(49:1) to give 2 (13.8 mg) and 4 (6.9 mg). Fraction I-G (3.6 g)
was subjected to ODS silica gel column chromatography using
MeOH–H2O (4:1) to afford four fractions (I-G1–4). Column chro-
matography of fraction I-G1 (912 mg) on silica gel and elution
with a stepwise gradient mixture of CHCl3–MeOH (100:1; 50:1;
20:1), and finally with MeOH alone, gave four fractions (I-G1a–
d). Fraction I-G1b (178 mg) was subjected to ODS silica gel col-
umn chromatography using MeCN–H2O (1:1) to give a mixture
of 11 and 15. The mixture was separated by preparative HPLC
using MeOH–H2O (7:3) to furnish 11 (5.4 mg, tR 26 min) and
15 (5.8 mg, tR 22 min). Fraction I-G1c (143 mg) was subjected
to ODS silica gel column chromatography using MeCN–H2O
(2:3) to give three fractions (fraction I-G1c1–3). Compound 12
(17.3 mg, 22 min) was obtained from fraction I-G1c1 (41.0 mg)
by subjecting it to preparative HPLC with MeCN–H2O (7:3). Puri-
fication of fractions I-G1c2 (16.6 mg) and I-G1c3 (36.5 mg) by
preparative HPLC using MeCN–H2O (1:1) led to the isolation of
5 (18.5 mg, 33 min) and 1 (8.0 mg, 41 min), respectively. Column
chromatography of fraction I-G2 (1.44 g) on silica gel and elution
with a stepwise gradient mixture of CHCl3–MeOH (30:1; 9:1),
and finally with MeOH alone, gave nine fractions (fraction I-
G2a–i). Fraction I-G2b (185 mg) was divided by ODS silica gel
column chromatography using MeOH–H2O (4:1) into two frac-
tions (I-G2b1 and I-G2b2). Fraction I-G2b1 (43.7 mg) was sub-
mitted to preparative HPLC using MeCN–H2O (1:1) to give 36
(8.8 mg, 52 min). Fraction I-G2b2 (351 mg) was purified by pre-
parative HPLC using MeOH–H2O (4:1) to give 20 (193 mg,
39 min).
5.3.1. Compound 1
Yellow powder; UV (MeOH) kmax (log e) 248 (4.15), 366 (4.40);
IR (film) mmax 3427 (OH), 1625 (C@O), 1595, 1507 and 1469 (aro-
matic ring); 1H NMR (acetone-d6, 500 MHz), see Table 1; 13C
NMR (acetone-d6, 125 MHz), see Table 2; HRESIMS m/z 371.1487
[M+H]+ (calcd for C21H23O6, 371.1495).
5.3.2. Compound 2
Yellow powder; UV (MeOH) kmax (log e) 383 (4.39); IR (film)
m
max 3375 (OH), 1702, 1614 (C@O), 1566 and 1494 (aromatic ring);
1H NMR (acetone-d6, 500 MHz), see Table 1; 13C NMR (acetone-d6,
125 MHz), see Table 2; HRESIMS m/z 409.2032 [M+H]+ (calcd for
C25H29O5, 409.2015).
5.3.3. Compound 3
Pale yellow powder; UV (MeOH) kmax (log e) 289 (4.09); IR
(film) mmax 3389 (OH), 1703, 1621 (C@O), 1496 and 1445 (aromatic
ring); 1H NMR (acetone-d6, 500 MHz), see Table 1; 13C NMR (ace-
tone-d6, 125 MHz), see Table 2; HRESIMS m/z 427.2133 [M+H]+
(calcd for C25H31O6, 427.2121).
5.3.4. Compound 4
Pale yellow powder; UV (MeOH) kmax (log e) 289 (4.09); IR
(film) mmax 3389 (OH), 1703, 1621 (C@O), 1496 and 1445 (aromatic
ring); 1H NMR (acetone-d6, 500 MHz), see Table 1; 13C NMR (ace-
tone-d6, 125 MHz), see Table 2; HRESIMS m/z 359.1499 [M+H]+
(calcd for C20H23O6, 359.1495).
5.3.5. Compound 5
Yellow powder; ½a D25
ꢂ
ꢁ10.0 (c 0.01, MeOH); CD [MeOH, nm,
De]:
) 318
221 (+2.41), 239 (ꢁ2.38), 290 (+1.13); UV (MeOH) kmax (log
e
(3.80), 280 (4.16); IR (film) mmax 3252 (OH), 1702, 1633 (C@O),
1506 and 1478 (aromatic ring); 1H NMR (acetone-d6, 500 MHz),
see Table 1; 13C NMR (acetone-d6, 125 MHz), see Table 2; HRESIMS
m/z 353.1407 [M+H]+ (calcd for C21H21O5, 353.1389).
Table 2
13C NMR spectral data of 1–10
Position
1
2
3
4
Position
5
6
7
8
9
10
70.4
62.8
33.6
130.1
109.0
152.3
109.8
149.8
112.8
118.5
126.2
78.8
1
2
3
4
130.7
113.1
144.6
148.3
128.2
122.8
121.0
148.7
138.7
149.0
112.0
119.6
120.5
138.4
188.0
28.6
115.7
164.6
113.9
162.1
107.5
129.6
126.8
113.3
145.1
146.8
129.1
123.4
117.8
145.2
192.5
21.8
111.2
163.8
115.7
162.6
107.9
130.1
128.5
114.5
144.5
142.0
128.1
121.9
73.6
42.1
204.9
21.8
122.6
131.2
25.4
111.1
163.9
115.7
162.7
107.9
130.2
129.6
117.0
145.1
144.0
115.3
121.2
73.6
41.9
204.9
21.7
122.6
131.2
25.4
2
3
4
5
6
7
8
9
70.0
31.8
30.4
70.4
32.0
30.8
84.6
73.4
152.2
124.1
175.4
116.2
127.4
148.7
145.7
145.7
117.7
125.3
130.6
113.8
159.9
113.8
130.6
28.4
74.4
74.5
192.7
129.3
111.2
165.4
103.1
164.1
112.5
128.9
130.0
128.1
155.8
115.0
127.1
28.7
189.9
129.6
111.5
159.6
109.5
157.4
113.9
117.5
156.8
102.7
161.6
105.3
128.3
115.6
128.9
77.9
129.7
109.0
152.4
110.0
150.0
114.5
121.5
163.6
102.7
163.6
115.4
133.9
117.1
129.2
75.6
129.7
108.7
152.2
109.9
150.2
115.1
118.8
156.3
103.1
157.5
107.3
128.2
118.7
126.1
78.8
5
6
10
20
30
10
40
10
50
20
60
30
a
b
40
67.8
22.5
50
C@O
100
200
300
400
500
1000
2000
3000
4000
5000
OMe
60
100
200
300
400
500
CHO
OMe
122.9
132.3
25.4
122.8
131.0
25.4
123.1
132.0
25.4
122.4
132.8
25.4
27.5
27.3
195.3
67.7
22.5
27.9
27.9
17.4
17.4
28.5
17.4
28.5
123.4
131.5
25.4
17.4
17.4
17.4
122.9
132.2
25.4
55.1
55.0
17.4
17.3
61.0

970
M. Kuroda et al. / Bioorg. Med. Chem. 18 (2010) 962–970
5.3.6. Formation of 5 from glabridin (20)
p4 ꢀ UASg-tk-luc were transfected into CV-1 cells. After 24 h of
transfection, the medium was changed to DMEM containing 10%
charcoal-treated FBS and each sample,36 and the cells were further
cultured for 24 h. Then, the cells were washed with Ca2+- and
Mg2+-containing phosphate-buffered saline (PBS+), to which Luc-
Lite (Perkin–Elmer, Wellesley, MA, USA) was added. The intensity
of emitted luminescence was determined using a TopCount micro-
plate scintillation/luminescence counter (Perkin–Elmer). The lumi-
nescence intensity ratio (test group/control group) was determined
A mixture of 20 (60.5 mg), dry aluminum chloride (24.1 mg),
ethyl orthoformate (3.1 mL), and dry benzene (23 mL) was stirred
at room temperature for 30 min after 5% hydrochloric acid
(70 mL) was added to it.34 The products were extracted with ethyl
ether and washed with water, dried and evaporated to dryness. The
residue was purified by preparative HPLC using MeCN–H2O (1:1)
to give 5 (1.2 mg, 34 min).
5.3.7. Compound 6
for each sample, and PPAR-c ligand-binding activity was expressed
as the relative luminescence intensity of the test sample to that of
the control sample.
Pale yellow powder; ½a D25
ꢂ
ꢁ23.0 (c 0.01, MeOH); CD [MeOH, nm,
D
e
]: 274 (ꢁ1.02), 293 (+4.88), 319 (ꢁ4.09), 358 (+0.98); UV
(MeOH) kmax (log e) 283 (4.09); IR (film) mmax 3357 (OH), 1520,
1474 (aromatic ring); 1H NMR (acetone-d6, 500 MHz), see Table 1;
13C NMR (acetone-d6, 25 MHz), see Table 2; HRESIMS m/z 341.1414
[M+H]+ (calcd for C20H21O5, 341.1389).
References and notes
1. Nicholas, S. B. Curr. Hypertens. Rep. 1999, 1, 131.
2. Unger, R. H. Annu. Rev. Med. 2002, 53, 319.
3. Burn, R. P.; Kim, J. B.; Hu, E.; Altiok, S.; Spiegelman, B. M. Curr. Opin. Cell Biol.
1996, 8, 826.
4. Morrison, R. F.; Farmer, S. R. J. Nutr. 2000, 130, 3116S.
5. Kaplan, F.; Al-Majali, K.; Betteridge, D. J. J. Cardiovasc. Risk 2001, 8, 211.
6. Moller, D. E. Nature 2001, 414, 821.
7. Mae, T.; Kishida, H.; Nishiyama, T.; Tsukagawa, M.; Konishi, E.; Kuroda, M.;
Mimaki, Y.; Sashida, Y.; Takahashi, K.; Kawada, T.; Nakagawa, K.; Kitahara, M. J.
Nutr. 2003, 133, 3369.
8. Kuroda, M.; Mimaki, Y.; Sashida, Y.; Mae, T.; Kishida, H.; Nishiyama, T.;
Tsukagawa, M.; Konishi, E.; Takahashi, K.; Kawada, T.; Nakagawa, K.; Kitahara,
M. Bioorg. Med. Chem. Lett. 2003, 13, 4267.
5.3.8. Compound 7
Pale yellow powder; ½a D25
ꢁ38.0 (c 0.01, MeOH); CD [MeOH, nm,
ꢂ
D
e
]: 210 (+3.81), 240 (+1.45), 304 (ꢁ4.01), 334 (+1.21); UV (MeOH)
kmax (log e) 313 (3.84), 276 (4.10); IR (film) mmax 3374 (OH), 1673
(C@O), 1608 and 1502 (aromatic ring); 1H NMR (acetone-d6,
500 MHz), see Table 1; 13C NMR (acetone-d6, 125 MHz), see Table 2;
HRESIMS m/z 341.1411 [M+H]+ (calcd for C20H21O5, 341.1389).
9. Nakagawa, K.; Kishida, H.; Arai, N.; Nishiyama, T.; Mae, T. Biol. Pharm. Bull.
2004, 27, 1775.
10. Aoko, F.; Honda, S.; Kishida, H.; Kitano, M.; Arai, N.; Tanaka, H.; Yokota, S.;
Nakagawa, K.; Asakura, T.; Nakai, Y.; Mae, T. Biosci. Biotechnol. Biochem. 2007,
71, 206.
11. Kajiyama, K.; Demizu, S.; Hiraga, Y.; Kinoshita, K.; Koyama, K.; Takahashi, K.;
Tamura, Y.; Okada, K.; Kinoshita, T. Phytochemistry 1992, 31, 3229.
12. Saitoh, T.; Shibata, S. Tetrahedron Lett. 1975, 4461.
13. Delle Monache, G.; De Rosa, M. C.; Scurria, R.; Vitali, A.; Cuteri, A.; Monacelli, B.;
Pasqua, G.; Botta, B. Phytochemistry 1995, 39, 575.
14. Kinoshita, T.; Kajiyama, K.; Hiraga, Y.; Takahashi, K.; Tamura, Y.; Mizutani, K.
Chem. Pharm. Bull. 1996, 44, 1218.
15. Ferrari, F.; Botta, B.; Alves de, L. R. Phytochemistry 1983, 22, 1663.
16. Fukai, T.; Sheng, C.-B.; Horikoshi, T.; Nomura, T. Phytochemistry 1996, 43, 1119.
17. Gottlieb, O. R.; Braga de, O. A.; Goncalves, T. M. M.; De Oliveira, G. G.; Pereira, S.
A. Phytochemistry 1975, 14, 2495.
18. Song, C.; Zheng, Z.; Liu, D.; Hu, Z. Zhiwu Xuebao 1997, 39, 486.
19. Saitoh, T.; Kinoshita, T.; Shibata, S. Chem. Pharm. Bull. 1976, 24, 752.
20. Mitscher, L. A.; Park, Y. H.; Omoto, S.; Clark, G. W.; Clark, D. Heterocycles 1978,
9, 1533.
5.3.9. Compound 8
Yellow powder; UV (MeOH) kmax (log e) 310 (4.06), 263 (3.57);
IR (film) mmax 3283 (OH), 2970, 2920, 1601 (C@O), 1512 and 1471
(aromatic ring); 1H NMR (acetone-d6, 500 MHz), see Table 1; 13C
NMR (acetone-d6, 125 MHz), see Table 2; HRESIMS m/z 353.1390
[M+H]+ (calcd for C21H21O5, 353.1390).
5.3.10. Compound 9
Yellow powder; UV (MeOH) kmax (log e) 296 (3.55); IR (film)
m
max 3374 (OH), 1673 (C@O), 1608, 1502, and 1463 (aromatic ring);
1H NMR (acetone-d6, 500 MHz), see Table 1; 13C NMR (acetone-d6,
125 MHz), see Table 2; HRESIMS m/z 369.1320 [M+H]+ (calcd for
C21H21O6, 369.1338).
5.3.11. Compound 10
21. Kinoshita, T.; Kajiyama, K.; Hiraga, Y.; Takahashi, K.; Tamura, Y.; Mizutani, K.
Heterocycles 1996, 43, 581.
22. Mitscher, L. A.; Park, Y. H.; Clark, D.; Beal, J. L. J. Nat. Prod. 1980, 43, 259.
23. Kinoshita, T.; Tamura, Y.; Mizutani, K. Chem. Pharm. Bull. 2005, 53, 847.
24. Kitagawa, I.; Chen, W. Z.; Hori, K.; Harada, E.; Yasuda, N.; Yoshikawa, M.; Ren, J.
Chem. Pharm. Bull. 1994, 42, 1056.
25. Asada, Y.; Li, W.; Yoshikawa, T. Phytochemistry 1998, 47, 389.
26. Fukui, H.; Goto, K.; Tabata, M. Chem. Pharm. Bull. 1988, 36, 4174.
27. Mizuno, M.; Tamura, K.; Tanaka, T.; Iinuma, M. Phytochemistry 1989, 28, 2811.
28. Pereira, M. O. D. S.; Fantine, E. C.; De Sousa, J. R. Phytochemistry 1982, 21, 488.
29. Fukai, T.; Wang, Q. H.; Takayama, M.; Nomura, T. Heterocycles 1990, 31, 373.
30. Kinoshita, T.; Saitoh, T.; Shibata, S. Chem. Pharm. Bull. 1976, 24, 991.
31. Nomura, T.; Fukai, T. ‘Phenolic Constituents of Licorice (Glycyrrhiza Species)’. In
‘Progress in the Chemistry of Organic Natural Products’; Herz, W., Kirby, G. W.,
Moore, R. E., Steglich, W., Tamm, C., Eds.; Springer Wien: New York, 1998; Vol.
73, p 27.
Pale yellow powder; UV (MeOH) kmax (log e) 283 (3.88); IR
(film) mmax 3376 (OH), 1608, 1478 and 1455 (aromatic ring); 1H
NMR (acetone-d6, 500 MHz), see Table 1; 13C NMR (acetone-d6,
125 MHz), see Table 2; HRESIMS m/z 249.1142 [M+H]+ (calcd for
C14H17O4, 249.1127).
5.4. PPAR-c ligand-binding activity
PPAR-
c ligand-binding activity was carried out using a GAL-4-
PPAR-
c
chimera assay system.35 CV-1 monkey kidney cells from
the American Type Culture Collection (ATCC) (Manassas, VA,
USA) were inoculated into a 96-well culture plate at 6 ꢀ 103
cells/well and incubated in 5% CO2/air at 37 °C for 24 h. As medium,
Dulbecco’s modified Eagle medium (DMEM) (Gibco, Grand Island,
NY, USA) containing 10% fetal bovine serum (FBS), 10 mL/L penicil-
lin-streptomycin (5000 IU/mL and 5000 lg/mL, Gibco), and 37 mg/
L ascorbic acid (Wako Pure Chemical, Tokyo, Japan) was used. Cells
were washed with OPTI-minimum essential medium (OPTI-MEM)
32. Gaffield, W. Tetrahedron 1970, 26, 4093.
33. Kiuchi, F.; Chen, X.; Tsuda, Y. Heterocycles 1990, 31, 629.
34. Fukai, T.; Nishizawa, J.; Yokoyama, M.; Tantai, L.; Nomura, T. Heterocycles 1994,
38, 1089.
35. Takahashi, N.; Kawada, T.; Goto, T.; Yamamoto, T.; Taimatsu, A.; Matsui, N.;
Kimura, K.; Saito, M.; Hosokawa, M.; Miyashita, K.; Fushiki, T. FEBS Lett. 2002,
514, 315.
36. The samples were dissolved in DMSO, to which the medium was added to
obtain the final concentration of 0.1% (v/v) of DMSO. DMSO was also added to
the control wells.
(Gibco) and transfected with pM-hPPAR-
using LipofectAMINE PLUS (Gibco). In a mock control, pM and
c
and p4 ꢀ UASg-tk-luc