A new flavan-3-ol and the anti-inflammatory effect of flavonoids from the fruit peels of Wisteria floribunda

Article abstract of DOI:10.1080/10286020.2011.603306

A new flavan-3-ol, (+)-afzelechin 5-O-β-D-glucopyranoside (2), together with 13 known flavonoids (1, 3-14), was isolated from the fruit peels of Wisteria floribunda. Their structures were assigned by detailed interpretation of NMR, MS, and CD spectroscopic data, as well as by comparing with published reports. The in vitro anti-inflammatory activity of the isolated compounds (1-14) was examined. Among them, compounds 3, 6, and 9 produced highest inhibitory effects on tumor necrosis factor alpha (TNF-α)-induced nuclear factor kappa-B activation in HepG2 cells with IC₅₀ values of 14.1, 16.5, and 11.9mM, respectively. With the exception of compound 6, the compounds significantly inhibited the accumulation of pro-inflammatory inducible nitric oxide synthase and cyclooxygenase-2 proteins in TNF-a-stimulated HepG2 cells at a concentration as low as 0.1 μM.

Full text of DOI:10.1080/10286020.2011.603306

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A new flavan-3-ol and the anti-
inflammatory effect of flavonoids from
the fruit peels of Wisteria floribunda
Bui Huu Tai a c , Trinh Nam Trung a b , Nguyen Xuan Nhiem a c , Do
Thi Ha a , Chu Van Men a b , Vu Binh Duong b , Hoang Van Luong b ,
SeokBean Song a , KiHwan Bae a & Young Ho Kim a
a College of Pharmacy, Chungnam National University , Daejeon,
305-764, Korea
b Vietnam Military Medical University , 104 Phung Hung Street,
Hadong District, Hanoi, Vietnam
c Vietnam Academy of Science and Technology , 18 Hoang Quoc
Viet Street, Hanoi, Vietnam
Published online: 10 Oct 2011.
To cite this article: Bui Huu Tai , Trinh Nam Trung , Nguyen Xuan Nhiem , Do Thi Ha , Chu
Van Men , Vu Binh Duong , Hoang Van Luong , SeokBean Song , KiHwan Bae & Young Ho Kim
(2011) A new flavan-3-ol and the anti-inflammatory effect of flavonoids from the fruit peels
of Wisteria floribunda , Journal of Asian Natural Products Research, 13:11, 1061-1068, DOI:
10.1080/10286020.2011.603306
To link to this article: http://dx.doi.org/10.1080/10286020.2011.603306
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Journal of Asian Natural Products Research
Vol. 13, No. 11, October 2011, 1061–1068
NOTE
A new flavan-3-ol and the anti-inflammatory effect of flavonoids from
the fruit peels of Wisteria floribunda
Bui Huu Taia,c†, Trinh Nam Trunga,b†, Nguyen Xuan Nhiema,c, Do Thi Haa,
Chu Van Mena,b, Vu Binh Duongb, Hoang Van Luongb, SeokBean Songa, KiHwan Baea
and Young Ho Kima*
aCollege of Pharmacy, Chungnam National University, Daejeon 305-764, Korea; bVietnam Military
Medical University, 104 Phung Hung Street, Hadong District, Hanoi, Vietnam; cVietnam Academy
of Science and Technology, 18 Hoang Quoc Viet Street, Hanoi, Vietnam
(Received 22 April 2011; final version received 3 July 2011)
A new flavan-3-ol, (þ)-afzelechin 5-O-b-D-glucopyranoside (2), together with 13
known flavonoids (1, 3–14), was isolated from the fruit peels of Wisteria floribunda.
Their structures were assigned by detailed interpretation of NMR, MS, and CD
spectroscopic data, as well as by comparing with published reports. The in vitro
anti-inflammatory activity of the isolated compounds (1–14) was examined. Among
them, compounds 3, 6, and 9 produced highest inhibitory effects on tumor necrosis
factor alpha (TNF-a)-induced nuclear factor kappa-B activation in HepG2 cells with
IC50 values of 14.1, 16.5, and 11.9 mM, respectively. With the exception of compound
6, the compounds significantly inhibited the accumulation of pro-inflammatory
inducible nitric oxide synthase and cyclooxygenase-2 proteins in TNF-a-stimulated
HepG2 cells at a concentration as low as 0.1 mM.
Keywords: COX-2; flavonoids; iNOS; NF-kB; Wisteria floribunda
1. Introduction
oxide and prostaglandins, via the pro-
inflammatory enzymes such as inducible
nitric oxide synthase (iNOS) and cycloox-
ygenase-2 (COX-2), have important roles
in tissue response to injury or infectious
agents [3]. These inducible enzymes are
essential components of the inflammatory
response, the ultimate repair of injury, and
carcinogenesis [3]. NF-kB regulates both
innate and adaptive immune responses. It is
activated rapidly in response to a wide
range of stimuli including pathogens,
stress signals, and pro-inflammatory cyto-
kines, such as TNF and IL-1 [4,5]. Thus,
agents that can suppress NF-kB activation
Persistent nuclear factor kappa-B (NF-kB)
activation is found in many chronic
inflammatory diseases, including rheuma-
toid arthritis (RA), asthma, inflammatory
bowel disease, ulcerative colitis, and
atherosclerosis, and plays a pivotal role in
these processes [1,2]. NF-kB controls the
expression of the cytokines including
interleukin-1 beta (IL-1b) and tumor
necrosis factor alpha (TNF-a), which are
the essential mediators of chronic inflam-
mation and implicated in leukocytosis,
hyperplasia, and tissue breakdown. It has
been demonstrated that high levels of nitric
*Corresponding author. Email: yhk@cnu.ac.kr
†These authors contributed equally to this work.
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2011 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2011.603306
http://www.tandfonline.com

1062
B.H. Tai et al.
have also the potential to suppress
inflammation [6].
2. Results and discussion
Dried fruit peels of W. floribunda were
extracted with 95% MeOH and fractio-
nated with n-hexane, ethyl acetate
(EtOAc), and n-butanol. During the
screening of plant extracts to identify
compounds that have inhibitory effects on
TNF-a-induced NF-kB activation in
human hepatocellular liver carcinoma
cell line (HepG2), the EtOAc extracts of
the fruit peels of W. floribunda showed
significant positive activity. To identify
active components, the EtOAc extracts
were isolated. The compounds isolated
and characterized contained a new flavan-
3-ol glycoside (2) and 13 known flavonoid
compounds (1, 3–14) (Figure 1).
Wisteria floribunda (Leguminosae) is a
woody liana cultivated widely in Korea,
Japan, the USA, and China. Its gall extracts
are used as an antitumoral preparation in
traditional Oriental medicine [7]. Many
Oriental medicine practitioners use W.
floribunda gall extracts for the treatment of
patients suffering from breast cancer,
stomach cancer, or RA. Extracts and
compounds isolated from the flowers and
seeds of W. floribunda have also been
reported to have antioxidant, anticancer,
and immunological activities [7,8]. In our
study, to identify anti-inflammatory lead
compounds from medicinal plants, we
extracted flavonoids from the fruit peels
of W. floribunda and studied their effects
on TNF-a-induced NF-kB activation and
their role in the accumulation of pro-
inflammatory iNOS and COX-2 proteins.
Compound 2 was obtained as light
brown solid with m.p. 215–2168C. The
UV spectrum showed an absorption
maximum at 276 nm, and the IR spectrum
exhibited absorption bands at 3392 (OH),
R1
OH
OH
OH
OH
O
HO
HO
HO
O
OH
OH
OR2
OH
7
R1
H
R2
H
1
2
3
OR1
H
Glc
H
OH
R2O
O
O
R1
OR3
OH
OH
R2O
O
R1
H
H
H
H
H
H
R2
R3
H
OH
8
H
OH
9
H
Glc
10
11
12
13
14
H
Api(1-6)Glc
R1
H
R2
H
H
Rha(1-6)Glc
4
5
6
Glc
Glc
H
H
H
Glc
H
Glc
Glc
OH
Glc
Figure 1. Structures of isolated compounds 1–14.

Journal of Asian Natural Products Research
1614, 1518, 1455 (CvC), 1244, and
1063
OH
1072 (CZOZC) cm21, which are the
characteristics of phenolic compounds.
Its positive electrospray ionization
(ESI)-MS displayed quasimolecular ions
at m/z 437 [M þ H]þ and m/z 459
[M þ Na]þ, corresponding to a molecular
formula of C21H24O10, which was further
confirmed by a high-resolution ESI-MS
(m/z 459.1241 [M þ Na]þ). The 13C NMR
and distortionless enhancement by polar-
ization transfer spectral data of 2 showed
21 carbons including 2 methylenes, 13
methines, and 6 quaternary olefinic car-
bons. Among them, one anomeric carbon
at dC 102.6, four oxygenated methine
carbons at dC 71.4, 75.0, 78.2, and 78.3,
and one oxygenated methylene carbon at
dC 62.6 were assigned to the sugar moiety.
Fifteen remaining carbons belonged to the
aglycone moiety. These data suggested
that 2 was a flavonoid glycoside. The
presence of three aglycone carbon signals
in the upfield at dC 83.0, 68.8, and 28.9
suggested that 2 belonged to the class of
flavan-3-ols. The 1H NMR spectrum
showed a pair of two-proton doublets at
dH 7.21 (2H, d, J ¼ 8.5 Hz, H-20,60) and dH
6.79 (2H, d, J ¼ 8.5 Hz, H-30,50) due to p-
substituted B-ring. Next, the absolute
configuration of 2 was determined by
careful examination of its 1D, 2D NMR,
and CD spectra. A negatively signed
maximum (D1 ¼ 29.1) at 275 nm in the
CD spectrum of 2 and, compared with a
CD empirical rule on the absolute
configuration at C-2 of flavan-3-ols (the
R-configuration in a negative Cotton curve
at 250–300 nm) [9], demonstrated the
R-configuration at C-2 of 2. The large
coupling constant value of H-2 (dH 4.61, d,
J2,3 ¼ 8.0 Hz) was consistent with the 2,3-
trans-configuration, compared with cate-
chin (2,3-trans, H-2, d, J2,3 ¼ 6.7 Hz) [10]
and epicatechin (2,3-cis, H-2, s) [10],
suggesting the S-configuration of C-3. In
addition, the 2R,3S-configuration of 2 was
also confirmed by 13C NMR spectrum. The
chemical shifts of C-2 (dC 83.0), C-3 (dC
4′
1′
O
HO
2
3
7
5
OH
4
HO
HO
HO
O
O
1″
OH
Figure 2. Key HMBC correlations of
compound 2.
68.8), and C-4 (dC 28.9) were similar to
those of 2R,3S-catechin 5-O-b-glucopyr-
anoside (C-2 at dC 82.9, C-3 at dC 68.7,
and C-4 at dC 28.4) [11] and different from
those of 2R,3R-epicatechin-5-O-b-D-glu-
copyranoside (C-2 at dC 78.2, C-3 at dC
64.7, and C-4 at dC 28.2) [12]. The
chemical shifts of C-5 (dC 158.0) and C-7
(dC 158.1) when compared with those of
catechin 5-O-b-glucopyranoside (C-5 at
dC 158.0, C-7 at dC 158.1) and catechin 7-
O-b-glucopyranoside (C-5 at dC 157.5, C-
7 dC at 158.7) [11] indicated that the sugar
moiety was linked to C-5 of aglycone. This
was also confirmed by the correlation
between the anomeric proton (dH 4.84) and
C-5 (dC 158.0) observed in the HMBC
spectrum (Figure 2). Finally, the presence
of a D-glucose unit was further confirmed
by acid hydrolysis and gas chromatog-
raphy (GC) analysis, followed by compar-
ing the retention times with authentic
samples (see Experimental). Based on the
above data, compound 2 was determined
as a new flavan-3-ol, (þ)-afzelechin 5-O-
b-D-glucopyranoside.
The other 13 compounds were charac-
terized as (þ)-afzelechin (1) [13], (þ)-
catechin (3) [10], (2)-epiafzelechin (4)
[14], (2)-epiafzelechin 7-O-b-D-gluco-
pyranoside (5) [15], (2)-epicatechin (6)
[10], dulcisflavan (7) [16], kaempferol (8)
[17], kaempferol 3-O-b-D-glucopyrano-
side (9) [18], kaempferol 3-O-b-D-apio-
furanosyl(1-6)-b-D-glucopyranoside (10)
[19], kaempferol 3-O-rutinoside (11)
[20], kaempferol 7-O-b-D-glucopyrano-
side (12) [21], kaempferol 3,7-di-O-b-D-

1064
B.H. Tai et al.
3
6
9
Compound
(µM)
0.1
+
1
+
10
+
0.1
+
1
+
10
+
0.1
+
1
+
10
+
–
+
–
+
–
+
TNF-α (10 ng/mL)
iNOS
Cox-2
GAPDH
Figure 3. Evaluation of TNF-a-induced iNOS and COX-2 mRNA expression in HepG2 cells.
glucopyranoside (13) [22], and kaempferol
3,40-di-O-b-D-glucopyranoside (14) [23]
(Figure 1). Their structures were
established on the basis of spectral and
chemical evidence, which were in close
agreement with those reported in the
literature.
HepG2 cells using concentrations of 1 and
10 mM. Lower band intensities were
observed compared with the stimulus
sample. Moreover, compounds 3 and 9
had inhibitory effects at a concentration
of 0.1 mM (Figure 3), indicating that
(þ)-catechin (3), (2)-epicatechin (6), and
kaempferol 3-O-b-D-glucopyranoside (9)
are the most active components. Among
them, compound 9, a major component of
the EtOAc extract (10.3% in dried EtOAc
extract weight), may cause major inhibitory
effects.
Anti-inflammatory activity of the iso-
lated compounds (1–14) was examined
in vitro at concentrations of 0–100 mM. As
shown in Table 2, compounds 3, 6, and 9
significantly inhibited the activation of NF-
kB induced by TNF-a in HepG2 cells. The
IC50 values were 11.9, 16.5, and 14.1mM,
respectively, in comparison with pyrroli-
dine dithiocarbamate as a positive control
(IC50 4.9 mM). Compounds 1 and 8
exhibited a moderate inhibitory effect.
The remaining compounds had weak
inhibitory effects. Up to concentration of
100 mM, these compounds did not show
cytotoxic effects by 3-(4,5-dimethylthiazol-
2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium (MTS) assay,
suggesting that the anti-inflammatory
effects of compounds did not result from
cytotoxicity. Compounds 3, 6, and 9 were
subjected to further assays for investigating
their anti-inflammatory effects. The upre-
gulation of pro-inflammatory iNOS and
COX-2 proteins in TNF-a-stimulated
HepG2 cells was evaluated using reverse
transcription polymerase chain reaction
(RT-PCR). Results showed that all of the
compounds significantly inhibited the
accumulation of pro-inflammatory (iNOS
and COX-2) proteins in TNF-a-stimulated
3. Experimental
3.1 General experimental procedures
Melting points were determined on an
Electrothermal 9100 melting point appar-
atus. Optical rotations were measured on a
JASCO DIP-370 automatic digital polari-
meter (JASCO, Tokyo, Japan). UV spectra
were acquired in MeOH using a JASCO V-
550 UV–VIS spectrometer. IR spectra
were recorded on a JASCO 100 IR
spectrophotometer. CD spectrum was
recorded on a JASCO J-725 spectrometer.
The NMR spectra were recorded using a
JEOL ECA-600 spectrometer (1H,
600 MHz; 13C, 150 MHz; JEOL, Tokyo,
Japan). The ESI mass spectra were
recorded on an Agilent 1100 LC-MSD
trap spectrometer (Agilent Technologies,
Palo Alto, CA, USA), and high-resolution
ESI spectra were obtained from a JEOL
JMS-HX/HX 110A tandem mass spec-
trometer (JEOL). GC spectra were

Journal of Asian Natural Products Research
1065
recorded on a Shimadzu-2010 spec-
trometer (Shimadzu, Kyoto, Japan). Silica
gel (70–230, 230–400 mesh, Merck,
Whitehouse Station, NJ, USA) and YMC
RP-18 resins (75 mm, Fuji Silysia Chemi-
cal Ltd, Kasugai, Japan) were used as
absorbents in the column chromatography.
Thin layer chromatography (TLC) plates
(silica gel 60 F254 and RP-18 F254, 0.25
mm, Merck) were purchased from Merck
KGaA. Spots were detected under UV
radiation (254 and 365 nm) and by
spraying the plates with 10% H2SO4
followed by heating with a heat gun.
pound 1 (34 mg) and compound 4 (20 mg).
Silica gel column chromatography of
fraction E3 (11.2 g) eluted with CHCl3–
MeOH–H2O (4/1/0.1, v/v/v) gave four
sub-fractions E3A–E3D. Sub-fractions
E3A (0.4 g) and E3B (0.3 g) were further
purified chromatographically, on silica gel,
eluting with (CH3)2CO/CHCl3/H2O
(2.5/1/0.1, v/v/v) to yield compounds 3
(34 mg) and 6 (133 mg). Compounds 5
(34 mg), 7 (50 mg), and 12 (12 mg) were
isolated from the sub-fraction E3D (0.8 g)
using YMC gel column chromatography
and eluted with (CH3)2CO/H2O (1/2.5).
Fraction E4 (32.7 g) appeared as precipi-
tates, from which the solvent was then
removed and the residue was washed
several times with warm methanol yielding
compound 9 (12.0 g). The remaining
solution was subjected to silica gel and
further to YMC gel chromatography to give
compound 2 (7 mg). Fraction E5 (1.4 g)
was chromatographed on a silica gel
column eluting with a solvent system of
CH2Cl2/CH3OH/H2O (4/1/0.1, v/v/v) to
obtain compounds 10 (7 mg) and 11 (6 mg).
Finally, compounds 13 (5 mg) and 14
(24 mg) were isolated from fraction
E6 (2.5 g) using a silica gel column
chromatography and further purified
by preparative TLC, eluted with (CH3)2-
CO/CHCl3/H2O (5/1/0.2, v/v/v). The
purity of isolated compounds was more
than 95%, quantified by analytical HPLC.
3.2 Plant material
The fruit peels of W. floribunda were
collected in Daejeon, Korea, in February
2009 and identified by Prof. KiHwan Bae,
College of Pharmacy, Chungnam
National University. A voucher specimen
(CNU 1389–5) is deposited in the
College of Pharmacy, Chungnam National
University.
3.3 Extraction and isolation
Dried sample (6 kg) was pulverized and
extracted with 95% methanol (18 l) under
reflux for 3 £ 3 h. The extract was con-
centrated to dryness under reduced press-
ure. The residue (496 g) was suspended in
water and partitioned, sequentially, with
n-hexane, ethyl acetate (EtOAc), and
n-butanol (BuOH) to yield n-hexane
extract (22.5 g), EtOAc extract (116 g),
and BuOH extract (210 g), respectively.
The EtOAc-soluble extract was fractioned
on a silica gel column and eluted with a
gradient solvent system of CH2Cl2 and
CH3OH (0–100% MeOH in volume) to
give six fractions E1–E6. Fraction E1
(2.6 g) was repeatedly chromatographed on
a silica gel column eluting with CHCl3–
CH3OH (10/1, v/v) to purify compound 8
(23 mg). The solution was further purified
using YMC gel column, eluting with
CH3OH–H2O (1/3, v/v) to obtain com-
3.3.1 Compound 2
Light brown solid, m.p. 215–2168C,
18
C21H24O10, ½aꢀD ¼ þ23.98 (c ¼ 0.45,
MeOH). UV (MeOH), lmax 276, 224 nm.
IR (KBr, cm21) 3392, 1614, 1518, 1455,
1
1244, and 1072. H NMR (methanol-d4,
600 MHz) and 13C NMR (methanol-d4,
150 MHz) spectral data, see Table 1.
ESI-MS (positive mode) m/z: 459
[M þ Na]þ, 437 [M þ H]þ, 275
[M þ H 2 162]þ. HR-ESI-MS (positive
mode) m/z: 459.1241 [M þ Na]þ (calcd
for C21H24O10Na 459.1267).

1066
B.H. Tai et al.
1
Table 1. The H and 13C NMR spectral data
for 2 in CD3OD.
column SPB-1 (0.25 mm £ 30 m); detec-
tor, FID; column temperature, 2108C;
injector temperature, 2708C; detector
temperature, 3008C; and carrier gas, He.
The absolute configuration of the mono-
saccharide was confirmed to be D-glucose
by comparing the retention time of the
monosaccharide derivative (tR 14.11 min)
with that of authentic sugar derivative
samples prepared in the same manner
(D-glucose derivative tR 14.11 min,
L-glucose derivative tR 14.26 min).
Position
daH (mult, J in Hz)
dCb
2
3
4
4.61 (d, 8.0)
3.98 (m)
83.0
68.8
28.9
2.57 (dd, 16.5, 8.5)
3.06 (dd, 16.5, 5.5)
5
6
–
6.29 (d, 2.2)
158.0
98.1
7
8
–
6.03 (d, 2.2)
158.1
96.9
9
10
–
–
–
156.8
103.5
131.4
129.7
116.2
158.5
10
20, 60
7.21 (d, 8.5)
6.79 (d, 8.5)
–
30, 50
3.5 Cell culture
40
Human hepatocarcinoma HepG2 cells
were maintained at 378C and 5% CO2 in
Dulbecco’s Modified Eagle’s Medium
(Invitrogen, Carlsbad, CA, USA) contain-
ing 10% heat-inactivated fetal bovine
serum, 100 units/ml penicillin, and
10 mg/ml streptomycin.
50-0O-Glc
1
4.84 (d, 7.5)
3.42c
102.6
75.0
78.3
71.4
78.2
62.6
200
300
400
500
600
3.53c
3.53c
3.61c
3.91 d (11.5) 3.73 dd
(11.5, 5.0)
Assignments were done by HMQC, HMBC, and
a
1H–1H COSY experiments. Measured at 600 MHz,
3.6 Cytotoxicity assay
b150 MHz, coverlapped signals.
An MTS assay (Promega’s CellTiter 96w
AQueous One Solution cell proliferation
Assay) was used to analyze the effect of
compounds on cell viability (Table 2).
Cells were cultured overnight in a 96-well
plate (,1 £ 104 cells/well). Cell viability
was assessed after exposure to compounds
at concentrations of 10, 50, and 100 mM for
24 h. The number of viable cells was
assessed by determination of A490nm of the
dissolved formazan product after addition
of MTS for 30 min as described by the
manufacturer (Promega, Madison, WI,
USA).
3.4 Acid hydrolysis of 2
Compound 2 (2.0 mg) was dissolved in
1.0 N HCl (dioxane/H2O, 1:1, v/v, 1.0 ml)
and then heated to 808C in a water bath for
3 h. The acidic solution was neutralized
with silver carbonate, and the solvent was
thoroughly driven out under N2 gas
overnight. After extraction with ethyl
acetate, the aqueous layer was concen-
trated to dryness using N2 gas. The residue
was dissolved in 0.1 ml of dry pyridine,
and then L-cysteine methyl ester hydro-
chloride in pyridine (0.06 M, 0.1 ml) was
added to the solution. The reaction mixture
was heated at 608C for 2 h, and 0.1 ml of
trimethylsilylimidazole solution was
added, followed by heating at 608C for
1.5 h. The dried product was partitioned
with n-hexane and H2O (0.1 ml, each), and
the organic layer was analyzed by gas
liquid chromatography (GLC): column,
3.7 NFkB-luciferase assay
Cells were seeded at 5 £ 105 cells/ml
(1.5 ml) in a 6-well plate and grown for
24 h. All cells were transfected using
Lipofectamine LTX (Invitrogen) as
described by the manufacturer. Luciferase
activity was assayed using an LB 953
AutoLumat (EG&G Berthold, Nashua,

Journal of Asian Natural Products Research
1067
CTAC-30, iNOS antisense 50-CTCAGGG-
TCACGGCCATTG-30, COX-2 sense
50-GCCCAGCACTTCACGCATCAG-30,
COX-2 antisense 50-GACCAGGCACCA-
GACCAAAGACC-30, GAPDH sense
50-TGTTGCCATCAATGACCCCTT-30,
and GAPDH antisense 50-CTCCACGAC-
GTACTCAGCG-30. The specificity of
products generated by each set of primers
was examined using gel electrophoresis
and further confirmed by melting curve
analysis.
Table 2. Inhibition effects of compounds 1–
14 on TNF-a-induced NF-kB activation in
HepG2 cells.
Compounds
IC50 (mM)a
1
2
34.0 ^ 2.7
.100
3
4
5
11.9 ^ 0.6
.100
.100
6
7
16.5 ^ 0.6
.100
8
9
10
27.4 ^ 1.3
14.1 ^ 0.7
.100
11
12
.100
.100
.100
.100
4.9 ^ 1.0
Acknowledgements
13
14
This work was supported partly by Priority
Research Centers Program through the
National Research Foundation of Korea
(NRF) funded by the Ministry of Education,
Science, and Technology (2009-0093815),
Republic of Korea. We are grateful to the
Korean Basic Science Institute for spectro-
scopic measurements.
PDTCb
a IC50 values were derived from dose–response
curves. Data are means of three independent
experiments.
b Pyrrolidine dithiocarbamate (purity , 99%, Sigma,
Seoul, Korea): positive control.
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(Chungnam National University, Daejeon,
Korea).
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