Diarylheptanoids from the seeds of Alpinia katsumadai as heat shock factor 1 inducers

Article abstract of DOI:10.1021/np200355n

Seven new diarylheptanoids, (-)-(R)-4′-hydroxyyashabushiketol (1), (3S,5S)-alpinikatin (2), katsumain C (3), 7-epi-katsumain C (4), ent-alpinnanin B (5), ent-alpinnanin A (6), and ent-calyxin H (8), were isolated from the EtOAc extract of the seeds of Alpinia katsumadai together with three known compounds, alpinnanin B (7), epicalyxin H (9), and calyxin H (10). Each isomer mixture of 3 and 4, 5-7, and 8-10 was separated successfully by preparative HPLC using a chiral column. The three isomer mixtures (3 and 4, 5-7, 8-10) at 1 μM increased expression of heat shock factor 1 (HSF1) with fold increases of 1.438, 1.190, and 1.316, respectively, which was accompanied with increased expression of heat shock protein (HSP) 27 (1.403-, 1.250-, and 1.270-fold, respectively) and HSP70 (1.373-, 1.313-, and 1.229-fold, respectively) without cellular cytotoxicity, suggesting a possible application of these compounds as HSP inducers. Celastrol was used as a positive control of HSP induction, producing fold increases of 1.066 (HSF1), 1.216 (HSP27), and 1.371 (HSP70) at 1 μM. Compounds 1 and 2 did not affect the induction of HSF1 protein.

Full text of DOI:10.1021/np200355n

ARTICLE
pubs.acs.org/jnp
Diarylheptanoids from the Seeds of Alpinia katsumadai as Heat
Shock Factor 1 Inducers
Joo-Won Nam,† Ga-Young Kang,† Ah-Reum Han,† Dongho Lee,‡ Yun-Sil Lee,† and Eun-Kyoung Seo*,†
†The Center for Cell Signaling & Drug Discovery Research, College of Pharmacy, Ewha Womans University, Seoul 120-750, Korea
‡School of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Korea
S Supporting Information
b
ABSTRACT: Seven new diarylheptanoids, (ꢀ)-(R)-400-hydro-
xyyashabushiketol (1), (3S,5S)-alpinikatin (2), katsumain C
(3), 7-epi-katsumain C (4), ent-alpinnanin B (5), ent-alpinnanin
A (6), and ent-calyxin H (8), were isolated from the EtOAc
extract of the seeds of Alpinia katsumadai together with three
known compounds, alpinnanin B (7), epicalyxin H (9), and
calyxin H (10). Each isomer mixture of 3 and 4, 5ꢀ7, and 8ꢀ10
was separated successfully by preparative HPLC using a chiral
column. The three isomer mixtures (3 and 4, 5ꢀ7, 8ꢀ10) at 1 μM increased expression of heat shock factor 1 (HSF1) with fold
increases of 1.438, 1.190, and 1.316, respectively, which was accompanied with increased expression of heat shock protein (HSP) 27
(1.403-, 1.250-, and 1.270-fold, respectively) and HSP70 (1.373-, 1.313-, and 1.229-fold, respectively) without cellular cytotoxicity,
suggesting a possible application of these compounds as HSP inducers. Celastrol was used as a positive control of HSP induction,
producing fold increases of 1.066 (HSF1), 1.216 (HSP27), and 1.371 (HSP70) at 1 μM. Compounds 1 and 2 did not affect the
induction of HSF1 protein.
he seeds of Alpinia katsumadai Hayata (Zingiberaceae) have
T
been used as an antiemetic and for treatment of gastric
disorders in Oriental Medicine.1 Previous phytochemical inves-
tigations of this plant have resulted in the isolation of various
types of diarylheptanoids,2ꢀ5 kavalactones,3 flavonoids,2,4,6,7
stilbenes,6 monoterpenes,6,7 and sesquiterpenes.8 Some of these
compounds have antioxidant,9 antiemetic,10,11 antiviral,5 cytopro-
tective,12 or other biological effects.
Heat shock transcription factor 1 (HSF1) plays a key role in
the cellular response that leads to the expression of heat shock
protein (HSP) genes under stress conditions.13 HSPs have
cytoprotective effects in neurodegenerative diseases and in other
types of cellular damage.14,15 As a part of a collaborative project
directed toward the discovery of HSP-modulating agents from
natural products, isolates from the seeds of A. katsumadai were
evaluated for their effects on HSF1 protein expression and on its
transcriptional targets, HSP27 and HSP70. Seven new com-
pounds, 1ꢀ6 and 8, were isolated from the EtOAc extract of the
seeds of this plant together with three known compounds 7, 9,
and 10. The three known compounds have not been isolated
previously from this plant. We report herein the isolation and
structural elucidation of 1ꢀ6 and 8. Compounds 1 and 2 and
three isomer mixtures of 3 and 4, 5ꢀ7, and 8ꢀ10 were also
evaluated for their HSF1-inducing activities in an H460 system.
elemental formula C19H20O3Na. The IR spectrum showed
absorption bands at 3312 cmꢀ1 for one or more hydroxy groups
and at 1717 cmꢀ1 for conjugated carbonyl groups.16 The 1H and
13C NMR spectra exhibited resonances at δH 7.24/δC 129.3,
7.25/129.2, 7.15/126.5, 7.56/131.3, and 6.90/116.9 and δC
143.5, 127.3, and 160.8 for two substituted benzene rings. In
the 1H NMR spectrum of 1, the protons of an olefinic function-
ality appeared at δH 6.70 and 7.58 with a 16.4 Hz coupling
constant, indicating their trans-configuration.16
A
13C NMR
’ RESULTS AND DISCUSSION
Compound 1 showed a molecular ion peak at m/z 319.1310
Received: April 26, 2011
Published: September 26, 2011
[M + Na]+ in the HR-ESIMS, corresponding to the sodiated
r
Copyright
2011 American Chemical Society and
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dx.doi.org/10.1021/np200355n J. Nat. Prod. 2011, 74, 2109–2115
American Society of Pharmacognosy
|

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ARTICLE
Figure 1. Δδ (δS ꢀ δR) values of MTPA esters of 1 and 2.
resonance at δC 200.1 indicated the presence of a carbonyl group.
The DEPT spectrum demonstrated three methylene groups at
δC 32.6, 40.0, and 48.5, along with one methine at δC 68.1. These
NMR data were similar to those of the known compound
(ꢀ)-(R)-yashabushiketol,17,18 except for the resonances of the
aromatic systems. The 1H and 13C NMR resonances at δH 7.56
(d, J = 8.6 Hz)/δC 131.3 (C-200 and C-600), 6.90 (d, J = 8.6 Hz)/
116.9 (C-300 and C-500), and δC 127.3 (C-100) and 160.8 (C-400)
indicated the presence of a p-hydroxyphenyl ring in the structure
of 1 instead of the monosubstituted benzene ring in (ꢀ)-(R)-
yashabushiketol.17,18 The HMBC correlations of 1 showed three-
bond correlations of H-20 and H-60/C-1, H-2/C-10, H-7/C-200
and C-600, and H-6/C-100, so that rings A and B could be assigned
at C-1 and C-7, respectively. Further analysis of the DEPT,
COSY, NOESY, HSQC, and HMBC data allowed unambiguous
assignments for the 1H and 13C NMR resonances. The configur-
ation of 1 was determined by the Mosher ester procedure.19
Compound 1 was treated with (S)- and (R)-MTPA-Cl, affording
(R)- and (S)-MTPA ester derivatives (1r and 1s), respectively.
The absolute configuration at C-3 was R on the basis of the Δδ
(δS ꢀ δR) values presented in Figure 1. Thus, compound 1 was
elucidated as a new diarylheptanoid, (3R)-3-hydroxy-1-phenyl-
7-(4-hydroxyphenyl)-6E-hepten-5-one, namely, (ꢀ)-(R)-400-
hydroxyyashabushiketol.
Figure 2. (A) HPLC chromatograms of mixtures of 3 and 4, 5ꢀ7, and
8ꢀ10. (B) HPLC chromatograms of separated compounds 3ꢀ10
[column: ChiralPak IB (10 ꢁ 250 mm); mobile phase: n-hexaneꢀIPA,
7:3; detection: UV 365 nm].
Compound 2 showed a molecular ion peak at m/z 321.1463
[M + Na]+ in the HR-ESIMS, corresponding to the sodiated
elemental formula C19H22O3Na. The IR spectrum showed an
1
absorption band at 3302 cmꢀ1. The H and 13C NMR spectra
exhibited resonances at δH 7.00/δC 130.4, 6.67/116.1, 7.37/
127.5, 7.29/129.6, and 7.20/128.6 and δC 134.4, 156.3, and
138.4 for two substituted benzene rings. In the 1H NMR
spectrum, a trans-olefinic group appeared at δH 6.20 (dd, J =
15.8, 6.8 Hz, H-6) and 6.58 (d, J = 15.8 Hz, H-7). The DEPT data
revealed the presence of two methines at δC 70.1 and 72.3 and
three methylenes at δC 32.0, 41.1, and 45.3. These NMR data
were similar to those of a known compound, (3S,5S)-3,5-
dihydroxy-1,7-diphenyl-6E-heptene,2 except for the presence of
a hydroxy group at C-40 of ring A {δH 7.00 (d, J = 8.6 Hz), 6.67
(d, J = 8.6 Hz)} in 2. Rings A and B of 2 were assigned to C-1 and
C-7, respectively, on the basis of the HMBC correlations of H-20
and H-60/C-1, H-2/C-10, H-7/C-200 and C-600, and H-6/C-100.
Further analysis of the DEPT, COSY, NOESY, HSQC, and
HMBC data allowed unambiguous assignments for the 1H and
13C NMR resonances. The Mosher ester procedure19 was per-
formed to determine the absolute configurations at C-3 and C-5,
which contain secondary hydroxyls, i.e., a diol. The configura-
tions at both C-3 and C-5 in 2 were “S” according to the Δδ
values (Figure 1), which revealed the typical pattern of syn-acyclic
1,3-diols.20 Thus, the structure of 2 was assigned to a new
compound, (3S,5S)-3,5-dihydroxy-1-(4-hydroxyphenyl)-7-phenyl-
6E-heptene, namely, (3S,5S)-alpinikatin.
A stereoisomeric mixture of 3 and 4 was obtained using
various column chromatography procedures as described in the
Experimental Section. The stereoisomers 3 and 4 were effectively
separated by preparative HPLC using a chiral column (ChiralPak
IB; 5 μm, 250 mm ꢁ 10 mm i.d.) as shown in Figure 2.
Compound 3 showed a molecular ion peak at m/z 567.2389
[M + H]+ in the HR-ESIMS, corresponding to an elemental
formula of C35H35O7. The IR spectrum indicated the presence of
one or more hydroxy groups at 3223 cmꢀ1 and conjugated
carbonyl groups at 1616 cm .
ꢀ1 16 The 1H and 13C NMR spectra
revealed the presence of two sets of p-substituted benzene rings
at δH 6.99/δC 130.1, 6.72/115.8, 7.12/129.4, and 6.69/115.3
and δC 156.1, 134.2, and 135.7 and a monosubstituted benzene
ring at δH 7.73/δC 129.2, 7.45/129.9, and 7.44/130.9 and δC
136.6. Resonances for two sets of trans-olefinic functionalities
appeared at δH 5.64 (dt, J = 15.4, 6.7 Hz, H-5)/δC 128.5 (C-5),
6.39 (dd, J = 15.4, 8.6 Hz, H-6)/134.7 (C-6), 8.03 (d, J = 16.0 Hz,
H-8000)/128.9 (C-8000), and 7.76 (d, J = 16.0 Hz, H-9000)/142.4
(C-9000). The 1H NMR resonance at δH 3.97 (3H, s), which was
correlated to the 13C NMR resonance at δC 56.3 in the HSQC
spectrum, indicated the presence of an aromatic methoxy group.
The methoxy group was assigned at C-4000 due to the three-bond
connectivity between the methoxy protons at δH 3.97 and C-4000
in the HMBC spectrum. The NOESY correlation between the
2110
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Journal of Natural Products
ARTICLE
1
methoxy protons and H-5000 provided further evidence for the
position of the methoxy group in 3. In the 13C NMR spectrum, a
carbonyl carbon appeared at δC 193.4, hydrogen-bonded to the
hydroxy proton resonating at δH 14.74 in the 1H NMR spectrum
in acetone-d6. The placement of substituents of ring C was
deduced by the HMBC correlations of H-5000/C-1000, C-3000, C-4000,
C-6000; OH-2000/C-1000, C-2000, C-3000; and H-7/C-1000, C-2000, C-6000.
These NMR data were similar to those of the known compounds
katsumains A and B,3 except for the presence of a p-hydroxy
group in rings A and B in 3. The HMBC data of 3 showed three-
bond correlations of H-20 and H-60/C-1 and H-200 and H-600/C-7
so that rings A and B could be assigned at C-1 and C-7, respec-
tively. The three-bond connectivities of H-11000 and 15000/C-9000
and H-8000/C-10000 provided evidence for the position of ring D at
C-9000. The linkage between C-7 of the diarylhepanoid and C-1000
of the chalcone moiety was deduced by the HMBC cross-peaks
of H-7/C-2000, C-6000 and H-6/C-1000. Further analysis of the
COSY, NOESY, HSQC, and HMBC data allowed unambiguous
and HMBC data allowed unambiguous assignments for the H
and 13C NMR resonances. The gross structure of 5 was identical
to that of the known compound alpinnanin B (7),22 which was
also isolated from A. katsumadai for the first time in the present
study. The spectroscopic data of 5 were similar to those of
compound 7, suggesting that these two compounds are stereo-
isomers. Compound 5 was dextrorotatory, [α]25D +7.0 (c 0.05,
MeOH), and showed a triplet-like splitting pattern for H2-4 in the
1H NMR spectrum. The configuration at both C-3 and C-7 was
R.4,21 Therefore, the structure of 5 was elucidated as the new
stereoisomer (2E)-1-{2,4-dihydroxy-3-[(1R,2E,5R)-5-hydroxy-
1-(4-hydroxyphenyl)-7-phenyl-2-hepten-1-yl]-6-methoxyphenyl}
-3-phenyl-2-propen-1-one, namely, ent-alpinnanin B.
Compound 6 showed a molecular ion peak at m/z 551.2427
[M + H]+ in the HR-ESIMS, corresponding to an elemental
formula of C35H35O6. The 1H and 13C NMR spectra were similar
to those of 5, except for the splitting pattern of H2-4 in the 1H
NMR spectrum, suggesting that these two compounds are
stereoisomers. Further analysis of the COSY, NOESY, HSQC,
and HMBC data confirmed that the gross structure of 6 was the
same as 5. Compound 6 was dextrorotatory, [α]25D +5.8 (c 0.07,
MeOH), and showed a quartet-like splitting pattern for H2-4 in
the 1H NMR spectrum. The configurations at C-3 and C-7 were
“S” and “R”,4,21 respectively, and the structure of 6 was elucidated
as the new stereoisomer (2E)-1-{2,4-dihydroxy-3-[(1R,2E,5S)-
5-hydroxy-1-(4-hydroxyphenyl)-7-phenyl-2-hepten-1-yl]-6-methoxy-
phenyl}-3-phenyl-2-propen-1-one, namely, ent-alpinnanin A.
1
assignments for the H and 13C NMR resonances. The estab-
lished method was applied to determine the absolute configura-
tion at C-3 and C-7 by comparison of the proton splitting
patterns of H2-4 and the optical activity to those of epicalyxin
H (9) and calyxin H (10) as previously described.4,21 Compound
3 exhibited a specific rotation of [α]25D +9.4 (c 0.2, MeOH) and
1
showed a triplet-like splitting pattern for H2-4 in the H NMR
spectrum. Thus, the configuration at C-3 and C-7 was R and the
structure of 3 was elucidated as a new compound, (2E)-1-{2,4-
dihydroxy-3-[(1R,2E,5R)-5-hydroxy-1,7-bis(4-hydroxyphenyl)-
2-hepten-1-yl]-6-methoxyphenyl}-3-phenyl-2-propen-1-one,
namely, katsumain C.
A stereoisomeric mixture of 8ꢀ10 was obtained by various
column chromatography procedures as described in the Experi-
mental Section. Compounds 8ꢀ10 were separated by prepara-
tive HPLC using a chiral column (ChiralPak IB; 5 μm, 250 mm ꢁ
10 mm i.d.) as shown in Figure 2.
Compound 4 showed a molecular ion peak at m/z 567.2380
[M + H]+ in the HR-ESIMS, corresponding to an elemental
formula of C35H35O7. The 1H and 13C NMR spectra of 4 were
similar to those of 3, except for the splitting pattern of H2-4 in the
1H NMR spectrum, suggesting that compound 4 was a stereo-
isomer of 3. Further analysis of the COSY, NOESY, HSQC, and
HMBC data confirmed that the gross structure of 4 was the same
as3. Compound4 was levorotatory, [α]25D ꢀ10.1 (c 0.1, MeOH),
Compound 8 showed a molecular ion peak at m/z 567.2387
[M + H]+ in the HR-ESIMS, corresponding to an elemental
formula of C35H35O7. The 1H and 13C NMR data exhibited two
p-substituted benzene rings at δH 7.12/δC 129.4, 6.68/115.3,
7.62/131.3, and 6.92/116.8 and δC 135.8, 155.9, 128.1, and
160.7 together with a monosubstituted benzene ring at δH 7.17/
δC 129.3, 7.24/129.1, and 7.18/126.3 and δC 143.7. These data
were similar to those of 5, except for the presence of a p-hydroxy
group in ring D in 8. The p-substituted benzene ring was assigned
as ring D due to the HMBC correlations of H-11000 and H-15000/
C-9000 and H-12000 and H-14000/C-10000. Further analysis of the
COSY, NOESY, HSQC, and HMBC data allowed unambiguous
assignments for the 1H and 13C NMR resonances. The spectro-
scopic data of 8 were similar to those of compounds 9 and 10,
except for the optical activities or the splitting patterns of H2-4 in
1
and showed a quartet-like splitting pattern for H2-4 in the H
NMR spectrum. Thus, the configurations at C-3 and C-7 in 4
were “R” and “S”, respectively. As a result, the structure of 4 was
elucidated as a new compound, (2E)-1-{2,4-dihydroxy-3-
[(1S,2E,5R)-5-hydroxy-1,7-bis(4-hydroxyphenyl)-2-hepten-1-
yl]-6-methoxyphenyl}-3-phenyl-2-propen-1-one, namely, 7-epi-
katsumain C.
A stereoisomeric mixture of 5ꢀ7 was separated by various
column chromatography methods as described in the Experi-
mental Section. Compounds 5ꢀ7 were each purified by pre-
parative HPLC using a chiral column (ChiralPak IB; 5 μm,
250 mm ꢁ10 mm i.d.) as shown in Figure 2.
1
the H NMR spectrum, suggesting that these compounds are
stereoisomers. Compound 8 was dextrorotatory, [α]25 +22.5
D
(c 0.1, MeOH) and had a triplet-like splitting pattern for H2-4 in
the 1H NMR spectrum. Thus, the configuration at C-3 and C-7
was R,4,21 and the structure of 8 was elucidated as a new stereoi-
somer, (2E)-1-{2,4-dihydroxy-3-[(1R,2E,5R)-5-hydroxy-1-(4-hy-
droxyphenyl)-7-phenyl-2-hepten-1-yl]-6-methoxyphenyl}-3-(4-
hydroxyphenyl)-2-propen-1-one, namely, ent-calyxin H.
Compound 5 showed a molecular ion peak at m/z 551.2440
[M + H]+ in the HR-ESIMS, corresponding to an elemental
formula of C35H35O6. The 1H and 13C NMR spectra were similar
to those of compounds 3 and 4, except for the absence of a p-
1
hydroxy group in ring A in 5. The H and 13C NMR data
exhibited two monosubstituted benzene rings at δH 7.17/δC
129.3, 7.24/129.3, 7.18/126.4, 7.74/129.2, 7.45/129.9, and
7.44/131.0 and δC 143.8 and 136.7, together with a p-substituted
benzene ring at δH 7.12/δC 129.5 and 6.69/115.4 and δC 135.8
and 156.1. The p-substituted benzene ring was assigned as ring B
due to the HMBC correlations of H-200 and H-600/C-7 and H-300
and H-500/C-100. Further analysis of the COSY, NOESY, HSQC,
In order to evaluate whether the isolates had inductive effects
on HSF1 and its transcriptional targets, HSP27 and HSP70,
Western blotting was performed at 12 and 24 h after treatment of
NCI-H460 cells with the compounds (12 h data not shown). The
results showed that both HSP27 and HSP70 protein expression,
as well as HSF1 protein expression, was increased significantly by
the isomeric mixtures of compounds 3 and 4, 5ꢀ7, and 8ꢀ10,
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ARTICLE
Table 1. H NMR Data of Compounds 1ꢀ6 and 8a
position
1
1b
2c
3b
4b
5b
6b
8b
1
2.71 m
2.55 m
2.66 m
1.70 m
2.57 m
2.69 m
1.65 m
1.76 m
3.61 m
2.57 m
2.70 m
1.64 m
1.79 m
3.64 m
2.66 m
2.79 m
1.70 m
1.80 m
3.63 m
2.66 m
2.79 m
1.68 m
1.83 m
3.65 m
2.65 m
2.79 m
1.70 m
1.80 m
3.62 m
2.84 m
1.80 m
2
3
4.13 p (6.0)
2.84 d (6.0)
3.73 m
1.80 m
4
2.27 t-like (6.7)
2.27 q-like (6.8)
2.28 t-like (6.8)
2.29 q-like (6.8)
2.28 t-like (6.6)
5
4.43 q (6.8)
5.64 dt (15.4, 6.7)
5.65 dt (15.4, 6.8)
5.64 dt (15.2, 6.8)
5.66 dt (15.2, 6.8)
5.63 dt (15.2, 6.6)
6
6.70 d (16.4) 6.20 dd (15.8, 6.8) 6.39 dd (15.4, 8.6) 6.39 dd (15.4, 8.5) 6.38 dd (15.2, 8.4) 6.40 dd (15.2, 8.4) 6.38 dd (15.2, 8.4)
7
7.58 d (16.4) 6.58 d (15.8)
5.21 d (8.6)
6.99 d (8.6)
6.72 d (8.6)
5.21 d (8.5)
7.00 d (8.8)
6.74 d (8.8)
5.22 d (8.4)
7.17 d (7.2)
7.24 t (7.2)
7.18 m
5.22 d (8.4)
7.18 d (7.2)
7.25 t (7.2)
7.19 m
5.21 d (8.4)
7.17 d (7.0)
7.24 t (7.0)
7.18 m
20,60
30,50
40
7.24 m
7.00 d (8.6)
6.67 d (8.6)
7.25 m
7.15 m
200,6”
300,500
400
7.56 d (8.6)
6.90 d (8.6)
7.37 d (7.4)
7.29 t (7.4)
7.20 m
7.12 d (8.8)
6.69 d (8.8)
7.12 d (8.6)
6.69 d (8.6)
7.12 d (8.4)
6.69 d (8.4)
7.12 d (8.4)
6.69 d (8.4)
7.12 d (8.8)
6.68 d (8.8)
5000
6.21 s
6.24 s
6.19 s
6.18 s
6.21 s
8000
9000
8.03 d (16.0)
7.76 d (16.0)
7.73 d (7.4)
7.45 t (7.4)
7.44 m
8.03 d (15.8)
7.75 d (15.8)
7.73 d (7.6)
7.45 t (7.6)
7.44 m
8.03 d (15.6)
7.76 d (15.6)
7.74 d (7.0)
7.45 t (7.0)
7.44 m
8.03 d (15.6)
7.76 d (15.6)
7.74 d (7.2)
7.45 t (7.2)
7.44 m
7.90 d (15.4)
7.75 d (15.4)
7.62 d (8.8)
6.92 d (8.8)
11000,15000
12000,14000
13000
OCH3-4000
OH-2000
3.97 s
3.96 s
3.94 s
3.94 s
3.94 s
14.74 s
14.74 s
14.75 s
14.75 s
14.94 s
a TMS was used as an internal standard; chemical shifts (δ) are expressed in ppm; J values are given in parentheses. b Data were measured in acetone-d6 at
400 MHz. c Data were measured in methanol-d4 at 400 MHz.
compared to those in untreated control cells. Celastrol, an HSP
inducer and positive control,23 also increased the expressions of
HSP27 and HSP70; however, celastrol did not affect the expres-
sion of HSF1, which indicates that celastrol and our isolates act
through different mechanisms. The most effective sample was
the mixture of compounds 3 and 4 as shown in Table 3; com-
pounds 1 and 2 did not modulate the expression of HSP27,
HSP70, and HSF1. The active compound mixtures of 3 and 4,
5ꢀ7, and 8ꢀ10 did not exhibit any cytotoxicity to NCI-H460
cells (IC50 values >30 μM), while the anticancer drug Taxol and
celastrol showed cytotoxicity with IC50 values of 8 and 12.3 μM,
respectively (Table 3).
carried out on an Acme 9000 system (Young Lin, South Korea) using
YMC J’sphere ODS-H80 (4 μm, 250 mm ꢁ 20 mm i.d.) and ChiralPak
IB (5 μm, 250 mm ꢁ10 mm i.d.) columns.
Plant Material. The seeds of A. katsumadai were purchased from
Kyungdong Oriental Herbal market in Seoul, South Korea, in May 2010
and identified by Professor Je-hyun Lee (College of Oriental Medicine,
Dongguk University). A voucher specimen (no. EA299) was deposited
at the Natural Product Chemistry Laboratory, College of Pharmacy,
Ewha Womans University.
Extraction and Isolation. The seeds of A. katsumadai (5.4 kg)
were extracted with MeOH (3 ꢁ 9 L) overnight at room temperature.
The solvent was evaporated in vacuo to afford a concentrated MeOH
extract (788 g). This extract was suspended in distilled H2O and
successively fractionated with n-hexane, EtOAc, and n-BuOH. A portion
of the EtOAc extract (150 g) was separated by silica gel flash column
chromatography (CC; 230ꢀ400 mesh, 2 kg) using CH2Cl2ꢀMeOH
(95.5:0.5 to 9:1) as a gradient solvent system to afford 16 fractions
(F01ꢀF16). Fraction F11 (6.0 g), eluted with CH2Cl2ꢀMeOH (85:15),
was subjected to reversed-phase CC (100 g) with MeCNꢀH2O (5:5) as a
solvent system to yield 17 subfractions (F11.01ꢀF11.17). Subfraction F11.03
(130 mg) was chromatographed using Sephadex LH-20 with 100% MeOH
to give compound 2 (56.0 mg, 0.00104% w/w). Subfraction F11.06 (80 mg)
was subjected to silica gel CC (10 g) using gradient mixtures of n-
hexaneꢀEtOAc (9:1 f 7:3) to afford compound 1 (43.0 mg, 0.000796%
w/w). Fractions F11.10 (200 mg) and F11.13 (110 mg) were chromato-
graphed on preparative HPLC using an isocratic mixture of MeOHꢀ0.1%
formic acid in water (82:18, 2 mL/min) as a solvent system to afford isomer
mixtures of compounds 8ꢀ10 (tR 50.3 min, 32.0 mg, 0.000593% w/w) and
compounds 3 and 4 (tR 43.2 min, 7.8 mg, 0.000144% w/w), respectively.
Fraction F11.15 (520 mg) was subjected to silica gel CC (50 g) with a
gradient of n-hexaneꢀEtOAc (9:1 f 5:5) as a solvent system to afford 15
subfractions (F11.15.01ꢀF11.15.15). Fraction F11.15.10 (75 mg) was
’ EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were
measured with a P-1010 polarimeter (JASCO, Japan) at 25 °C. UV
and IR spectra were recorded on a U-3000 spectrophotometer (Hitachi,
Japan) and a FTS 135 FT-IR spectrometer (Bio-Rad, CA), respectively.
1D and 2D NMR experiments were performed on a UNITY INOVA
400 MHz FT-NMR instrument (Varian, CA) with tetramethylsilane
(TMS) as an internal standard. Mass spectrometry was carried out with a
Waters ACQUITY UPLC system coupled to a Micromass Q-Tof Micro
mass spectrometer and Agilent 6220 Accurate-Mass TOF LC/MS
system. Silica gel (230ꢀ400 mesh, Merck, Germany), RP-18 (YMC
gel ODS-A, 12 nm, S-150 μm), and Sephadex LH-20 (Amersham
Pharmacia Biotech) were used for column chromatography. TLC was
performed on Kieselgel 60 F 254 (silica gel, 0.25 mm layer thickness,
Merck, Germany) and RP-18 F 254s (Merck, Germany) plates, with
visualization under UV light (254 and 365 nm) and 10% (v/v) H2SO4
spray followed by heating (120 °C, 5 min). Preparative HPLC was
2112
dx.doi.org/10.1021/np200355n |J. Nat. Prod. 2011, 74, 2109–2115

Journal of Natural Products
ARTICLE
Table 2. 13C NMR Data of Compounds 1ꢀ6 and 8a
position
1b
2c
3b
4b
5b
6b
8b
1
2
3
4
5
6
7
32.6, CH2
40.0, CH2
68.1, CH
48.5, CH2
200.1, C
32.0, CH2
41.1, CH2
70.1, CH
45.3, CH2
72.3, CH
133.3, CH
131.5, CH
134.4, C
31.7, CH2
39.9, CH2
70.7, CH
41.7, CH2
128.5, CH
134.7, CH
43.0, CH
134.2, C
31.8, CH2
39.9, CH2
70.8, CH
41.7, CH2
128.4, CH
134.7, CH
42.9, CH
134.3, C
32.7, CH2
39.7, CH2
70.8, CH
41.8, CH2
128.5, CH
134.9, CH
43.1, CH
143.8, C
32.7, CH2
39.7, CH2
70.9, CH
41.8, CH2
128.4, CH
134.8, CH
43.0, CH
143.8, C
32.6, CH2
39.6, CH2
70.7, CH
41.6, CH2
128.3, CH
134.9, CH
43.0, CH
143.7, C
124.9, CH
143.6, CH
143.5, C
10
20,60
30,50
40
129.3, CH
129.2, CH
126.5, CH
127.3, C
130.4, CH
116.1, CH
156.3, C
130.1, CH
115.8, CH
156.1, C
130.1, CH
115.8, CH
156.1, C
129.3, CH
129.3, CH
126.4, CH
135.8, C
129.3, CH
129.3, CH
126.4, CH
135.9, C
129.3, CH
129.1, CH
126.3, CH
135.8, C
100
138.4, C
135.7, C
135.9, C
200,600
300,500
400
131.3, CH
116.9, CH
160.8, C
127.5, CH
129.6, CH
128.6, CH
129.4, CH
115.3, CH
156.1, C
129.3, CH
115.3, CH
156.1, C
129.5, CH
115.4, CH
156.1, C
129.4, CH
115.4, CH
156.1, C
129.4, CH
115.3, CH
155.9, C
1000
111.8, C
111.8, C
111.9, C
112.0, C
112.0, C
2000
166.5, C
166.5, C
165.9, C
166.5, C
166.5, C
3000
106.2, C
106.2, C
106.1, C
106.3, C
106.3, C
4000
162.3, C
162.3, C
162.4, C
162.4, C
162.2, C
5000
6000
92.3, CH
164.2, C
92.3, CH
164.0, C
92.3, CH
164.2, C
92.4, CH
163.9, C
92.1, CH
163.1, C
7000
193.4, C
193.4, C
193.5, C
193.5, C
193.4, C
8000
9000
128.9, CH
142.4, CH
136.6, C
128.9, CH
142.3, CH
136.6, C
129.0, CH
142.5, CH
136.7, C
128.9, CH
142.4, CH
136.6, C
125.5, CH
143.1, CH
128.1, C
10000
11000,15000
12000,14000
13000
OCH3-40000
129.2, CH
129.9, CH
130.9, CH
56.3, CH3
129.2, CH
129.9, CH
130.9, CH
56.3, CH3
129.2, CH
129.9, CH
131.0, CH
56.3, CH3
129.2, CH
129.9, CH
131.0, CH
56.3, CH3
131.3, CH
116.8, CH
160.7, C
56.2, CH3
a TMS was used as an internal standard; Chemical shifts (δ) are expressed in ppm. b Data were measured in acetone-d6 at 100 MHz. Data were
c
measured in methanol-d4 at 100 MHz.
chromatographed over reversed-phase CC (8 g) using an isocratic
solvent system of MeOHꢀH2O (85:15) to yield an isomer mixture of
compounds 5ꢀ7 (31.0 mg, 0.000574% w/w). The mixtures of com-
pounds 3 and 4, 5ꢀ7, and 8ꢀ10 were subjected to preparative HPLC
using a chiral selective column with n-hexaneꢀIPA (7:3, 1 mL/min) as
the solvent system to provide 3 (0.90 mg, tR 41.1 min, 0.000017% w/w),
4 (2.00 mg, tR 52.4 min, 0.000037% w/w), 5 (2.10 mg, tR 36.7 min,
0.000039% w/w), 6 (2.20 mg, tR 43.1 min, 0.000041% w/w), 7 (1.30 mg,
tR 46.9 min, 0.000024% w/w), 8 (1.10 mg, tR 38.5 min, 0.000020% w/w),
9 (2.00 mg, tR 44.3 min, 0.000037% w/w), and 10 (2.30 mg, tR
48.3 min, 0.000043% w/w).
Western Blot Analysis. The ability of compounds isolated from A.
katsumadai to modulate HSF1 and HSPs expression was evaluated by
established protocol.24 Proteins in lysates were separated by SDS-PAGE,
electrotransferred to nitrocellulose membranes (GE Healthcare, UK),
subsequently blotted with the specified antibodies, and visualized with
an ECL detection system (Thermo Scientific, USA). Anti-HSF1,
-Hsp27, and -Hsp70 and β-actin antibodies were purchased from Santa
Cruz Biotechnology (USA).
NMR data, see Table 2; NOESY correlations H-1/H-20 and H-60, H-7/H-
200 and H-600; HMBC correlations H-40/C-30; H-30 and H-50/C-10; H-20
and H-60/C-1; H-1/C-10, C-20, C-60, C-2, C-3; H-2/C-10, C-1, C-3, C-4;
H-3/C-1, 0C0 -2, C-4, C-5; H-4/C-2, C-3, C-5; H-6/C-4, C-5, C-7, C-100,
C-200, C-6 ; H-7/C-5, C-6, C-100, C-200, C-600; H-200 and H-600/C-7, C-300,
C-400, C-500; H-300 and H-500/C-100, C-400; HR-ESIMS m/z 319.1310
[M + Na]+ (calcd for C19H20O3Na, 319.1305).
(3S,5S)-Alpinikatin (2): yellow powder; [α]25D +19.3 (c 0.1, MeOH);
UV (MeOH) λmax (log ε) 252 (4.4), 227 (4.2) nm; IR (KBr) νmax 3302,
2926, 1554, 1455 cmꢀ1; 1H NMR data, see Table 1; 13C NMR data, see
Table 2; NOESY correlations H-1/H-20 and H-60, H-3/H-5, H-7/H-200
and H-600; HMBC correlations H-30 and H-50/C-10, C-40; H-20 and
H-60/C-30, C-40, C-50, C-1; H-1/C-10, C-20, C-60, C-2, C-3; H-2/C-10,
C-1, C-4; H-3/C-1, C-2, C-4, C-5; H-4/C-2, C-3, C-5, C-6; H-5/C-3,
C-4, C-6, 0C0 -7; H-6/C-4, C-5, C-100, C-200, C-600; H-7/C-5, C-6, C-100,
C-200, C-6 ; H-200 and H-600/C-7, C-100, C-400; H-300 and H-500/C-100,
C-200, C-600; H-400/C-200, C-300, C-500, C-600; HR-ESIMS m/z 321.1463
[M + Na]+ (calcd for C19H22O3Na, 321.1461).
Katsumain C (3): yellow, amorphous solid; [α]25 +9.4 (c 0.2,
D
MTT Assay. The cells were assayed for their cytotoxicity in the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT; Sigma)
test according to established protocol.25
MeOH); UV (MeOH) λmax (log ε) 348 (4.7), 288 (4.5), 217 (4.9) nm;
IR (KBr) νmax 3223, 1616, 1559, 1509, 1454 cmꢀ1; 1H NMR data, see
Table 1; 13C NMR data, see Table 2; NOESY correlations H-1/H-20 and
H-60, H-5/H-7, H-7/H-200 and H-600, OCH3ꢀ4000/H-5000, H-9000/H-11000
and H-15000; HMBC correlations H-30 and H-50/ C-10, C-40; H-20 and
H-60/C-30, C-40, C-50, C-1; H-1/C-10, C-20, C-60, C-2, C-3; H-2/C-10,
(ꢀ)-(R)-400-Hydroxyyashabushiketol (1): yellow powder; [α]25D ꢀ18.5
(c 0.1, MeOH); UV (MeOH) λmax (log ε) 325 (4.7), 233 (4.6) nm; IR
(KBr) νmax 3312, 2929, 1717, 1541 cmꢀ1; 1H NMR data, see Table 1; 13
C
2113
dx.doi.org/10.1021/np200355n |J. Nat. Prod. 2011, 74, 2109–2115

Journal of Natural Products
ARTICLE
Table 3. Induction of HSF1 and HSPs by Isolates from A.
katsumadai
H-20 and H-60/C-40, C-1; H-1/C-10, C-20, C-60, C-2, C-3; H-2/C-10, C-1,
C-3, C-4; H-3/C-1, C-5; H-4/C-2, C-3, C-6; H-5/C-3, C-4, C-7; H-6/
C-4, C-7, C-100; H-7/C-6, C-200, C-600, C-1000, C-2000, C-6000; OH-2000/
C-1000, C-2000, C-3000; OCH3-4000/C-4000, C-5000; H-5000/C-1000, C-3000, C-4000,
C-6000, C-7000;00H0 -8000/C-7000, C-9000, C-10000; H-9000/C-7000, C-10000, C-11000,
C-15000; H-11 and H-15000/C-13000; H-12000 and H-14000/C-10000; H-13000/
C-12000, C-14000; HR-ESIMS m/z 551.2427 [M + H]+ (calcd for
C35H35O6, 551.2428).
fold increaseb
compound(s)
HSF1
HSP27
HSP70
IC50 (μM)c
3 and 4a
5ꢀ7a
1.438 ( 0.032 1.403 ( 0.023 1.373 ( 0.012
1.190 ( 0.005 1.250 ( 0.011 1.313 ( 0.047
1.316 ( 0.053 1.270 ( 0.031 1.229 ( 0.009
1.066 ( 0.009 1.216 ( 0.022 1.371 ( 0.037
47.4
39.8
45.7
12.3
8.0
Alpinnanin B (7): [α]25 ꢀ8.0 (c 0.1, MeOH); HR-ESIMS m/z
D
8ꢀ10a
celastrold
Taxol
551.2434 [M + H]+ (calcd for C35H35O6, 551.2428); physical and
spectroscopic data were comparable to literature values.22
NDe
NDe
NDe
Ent-calyxin H (8): yellow, amorphous solid; [α]25 +22.5 (c 0.1,
D
a Protein expressions were assayed after treatment with isomeric mix-
tures. b Quantitative immunoblotting results for HSF1, HSP27, and
HSP70 in NCI-H460 (human non-small-cell lung cancer cells) after
normalization to the β-actin signal are summarized. Data shown
represent the mean ( SD of three independent experiments performed
in triplicate at 24 h of treatment. Statistically significant difference (p <
0.05) in comparison with the quantitative value of HSP70, HSP27, or
MeOH); UV (MeOH) λmax (log ε) 371 (4.8), 228 (4.7) nm; IR (KBr)
νmax 3260, 1615, 1558, 1510, 1437 cmꢀ1; 1H NMR data, see Table 1;
13C NMR data, see Table 2; NOESY correlations H-1/H-20 and H-60,
H-7/H-200 and H-600, OCH3-4000/H-5000, H-9000/H-11000 and H-15000;
HMBC correlations H-40/C-30, C-50; H-30 and H-50/C-10; H-20 and
H-60/C-40, C-1; H-1/C-10, C-20, C-60, C-2, C-3; H-2/C-1, C-3; H-4/
C-2, C-3, C-5, C-6; H-5/C-4, C-7; H-6/C-4, C-7, C-1000; H-7/C-5, C-6,
C-100, C-200, C-600, C-1000, C-2000, C-6000; H-200 and H-600/C-7, C-400; H-300
and H-500/C-100, C-400; OCH3-4000/C-4000; H-5000/C-1000, C-3000, C-4000,
C-6000; H-8000/C-7000, C-9000, C-10000; H-9000/C-7000, C-8000, C-10000, C-11000,
C-15000; H-11000 and H-15000/C-9000, C-13000; H-12000 and H-14000/C-10000,
C-13000; HR-ESIMS m/z 567.2387 [M + H]+ (calcd for C35H35O7,
567.2377).
c
HSF1 level between treated and untreated control cells. IC50 values
were the concentrations (μM) necessary for 50% inhibition of cell
growth in NCI-H460 cells. d Celastrol was used as a positive control.
e ND; not detected
C-2, C-3; H-3/C-1, C-5; H-4/C-2, C-3, C-5, C-6; H-5/C-4, C-7; H-6/
C-4, C-7, C-100, C-1000; H-7/C-5, C-200, C-600, C-1000, C-2000, C-6000; H-200
and H-600/C-7, C-400; H-300 and H-500/C-100, C-400; OH-2000/C-1000, C-2000,
C-3000; OCH3-4000/C-4000; H-5000/C-1000, C-3000, C-4000, C-6000; H-8000/C-7000,
C-10000; H-9000/C-7000, C-8000, C-10000, C-11000, C-15000; H-11000 and H-15000/
C-9000, C-13000; H-12000 and H-14000/C-10000; H-13000/C-12000; HR-ESIMS
m/z 567.2389 [M + H]+ (calcd for C35H35O7, 567.2377).
Epicalyxin H (9): [α]25 +9.3 (c 0.09, MeOH); HR-ESIMS m/z
D
567.2382 [M + H]+ (calcd for C35H35O7, 567.2377); physical and
spectroscopic data were comparable to literature values.21
Calyxin H (10): [α]25 ꢀ14.9 (c 0.07, MeOH); HR-ESIMS m/z
D
567.2370 [M + H]+ (calcd for C35H35O7, 567.2377); physical and
spectroscopic data were comparable to literature values.21
7-epi-Katsumain C (4): yellow, amorphous solid; [α]25 ꢀ10.1 (c
Preparation of the (S)- and (R)-MTPA Ester Derivatives of 1
and 2 by the Mosher Ester Procedure. (S)- and (R)-MTPA esters
of compounds 1 and 2 were prepared using the Mosher ester pro-
cedure.19 Compounds 1 and 2 (1 mg each) were dried under vacuum,
resuspended in pyridine-d5 (1 mL each), and transferred into clean
NMR tubes, respectively. (S)-(+)-α-Methoxy-α-(trifluoromethyl)phe-
nylacetyl chloride [(S)-MTPA-Cl] (10 μL) and DMAP were immedi-
ately added into each NMR tube under an N2 gas stream, and then the
NMR tubes were shaken to ensure even mixing. The NMR tubes were
incubated in a water bath for 4 h (40 °C). The reactions afforded
(R)-MTPA ester derivatives 1r and 2r, respectively. In the same manner
except for the treatment with (R)-MTPA-Cl (10 μL), (S)-MTPA ester
derivatives (1s and 2s) were prepared. The spectra of 1r, 1s, 2r, and 2s
D
0.1, MeOH); UV (MeOH) λmax (log ε) 347 (4.7), 288 (4.5), 216
(4.8) nm; IR (KBr) νmax 3225, 1616, 1560, 1511, 1458 cmꢀ1; 1H NMR
data, see Table 1; 13C NMR data, see Table 2; NOESY correlations H-1/
H-20 and H-60, H-5/H-7, H-7/H-200 and H-600, OCH3ꢀ4000/H-5000,
H-9000/H-11000 and H-15000; HMBC correlations H-30 and H-50/C-10,
C-40; H-20 and H-60/ C-30, C-40, C-50, C-1; H-1/C-10, C-20, C-60, C-2,
C-3; H-2/C-1, C-3, C-4; H-3/C-1; H-4/C-2, C-3, C-5; H-5/C-3, C-4,
C-7; H-6/C-4, C-7, C-1000; H-7/C-5, C-6, C-100, C-200, C-600, C-1000, C-2000,
C-6000; H-200 and H-600/C-7, C-400; H-300 and H-500/C-100, C-400; OH-2000/
C-1000, C-2000, C-3000; OCH3-4000/C-4000; H-5000/C-1000, C-3000, C-4000, C-6000,
C-7000; H-8000/C-7000, C-10000; H-9000/C-7000, C-10000, C-11000, C-15000; H-11000
and H-15000/C-9000, C-13000; H-12000 and H-14000/C-10000; H-13000/C-12000;
HR-ESIMS m/z 567.2380 [M + H]+ (calcd for C35H35O7, 567.2377).
Ent-alpinnanin B (5): yellow, amorphous solid; [α]25D +7.0 (c 0.05,
MeOH); UV (MeOH) λmax (log ε) 350 (4.8), 289 (4.5), 217 (4.9) nm; IR
(KBr) νmax 3255, 1616, 1559, 1509, 1427 cmꢀ1;1HNMRdata, seeTable1;
13C NMR data, see Table 2; NOESY correlations H-1/H-20 and H-60, H-5/
H-7, H-7/H-200 and H-600, OCH3ꢀ4000/H-5000, H-9000/H-11000 and H-15000;
HMBC correlations H-40/C-30, C-50; H-30 and H-50/C-10; H-20 and H-60/
C-40, C-1; H-1/C-10, C-20, C-60, C-2, C-3; H-2/C-10, C-1, C-3, C-4; H-3/C-
1, C-5; H-4/C-2, C-3, C-6; H-5/C-3, C-4, C-7; H-6/C-4, C-7, C-100; H-7/
C-6, C-200, C-600, C-1000, C-2000, C-6000; OH-2000/C-1000, C-2000, C-3000; OCH3-
4000/C-4000, C-5000; H-5000/C-1000, C-3000, C-4000, C-6000, C-7000; H-8000/C-7000,
C-9000, C-10000; H-9000/C-7000, C-10000, C-11000, C-15000; H-11000 and H-15000/
C-13000; H-12000 and H-14000/C-10000; H-13000/C-12000, C-14000; HR-ESIMS m/
z 551.2440 [M + H]+ (calcd for C35H35O6, 551.2428).
1
were obtained directly from the reaction NMR tubes. H NMR data
(pyridine-d5, 400 MHz) for 1s: δ 6.954 (1H, d, J = 16.4 Hz, H-6), 6.109
(1H, m, H-3), 3.404 (1H, dd, J = 17.2, 8.0 Hz, H-4a), 3.156 (1H, dd, J =
17.2, 4.0 Hz, H-4b), 2.766 (2H, m, H-1), 2.200 (2H, m, H-2). 1r: δ 7.046
(1H, d, J = 16.4 Hz, H-6), 6.090 (1H, m, H-3), 3.450 (1H, dd, J = 17.2,
8.0 Hz, H-4a), 3.222 (1H, dd, J = 17.2, 4.0 Hz, H-4b), 2.696 (2H, m,
H-1), 2.126 (2H, m, H-2). 2s: δ 7.044 (1H, d, J = 16.0 Hz, H-7), 6.549
(1H, dd, J = 16.0, 8.0 Hz, H-6), 6.121 (1H, q, J = 8.0 Hz, H-5), 5.445
(1H, m, H-3), 2.558 (1H, m, H-4a), 2.549 (2H, m, H-1), 2.276 (1H, m,
H-4b), 2.008 (2H, m, H-2). 2r: δ 6.876 (1H, d, J = 15.8 Hz, H-7), 6.331
(1H, dd, J = 15.8, 7.4 Hz, H-6), 5.963 (1H, q, J = 7.4 Hz, H-5), 5.490
(1H, m, H-3), 2.764 (2H, m, H-1), 2.518 (1H, m, H-4a), 2.297 (1H, m,
H-4b), 2.192 (2H, m, H-2).
Ent-alpinnanin A (6): yellow, amorphous solid; [α]25D +5.8 (c 0.07,
MeOH); UV (MeOH) λmax (log ε) 351 (4.8), 289 (4.5), 217 (4.9) nm;
IR (KBr) νmax 3255, 1616, 1561, 1507, 1427 cmꢀ1; 1H NMR data, see
Table 1; 13C NMR data, see Table 2; NOESY correlations H-1/H-20 and
H-60, H-5/H-7, H-7/H-200 and H-600, OCH3-4000/H-5000, H-9000/H-11000
and H-15000; HMBC correlations H-40/C-30, C-50; H-30 and H-50/C-10;
’ ASSOCIATED CONTENT
S
Supporting Information. Spectroscopic data including
b
1H, 13C, DEPT, and 2D NMR of compounds 1 and 2; 1H, 13C,
1
and 2D NMR of compounds 3ꢀ6 and 8; and H NMR of the
2114
dx.doi.org/10.1021/np200355n |J. Nat. Prod. 2011, 74, 2109–2115

Journal of Natural Products
ARTICLE
(R)- and (S)-MTPA esters of compounds 1 and 2 are available
free of charge via the Internet at http://pubs.acs.org.
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’ AUTHOR INFORMATION
Corresponding Author
*Tel: +82-2-3277-3047. Fax: +82-2-3277-3051. E-mail: yuny@
ewha.ac.kr.
Karunagaran, D.; Anto, R. J. J. Biol. Chem. 2005, 280, 6301–6308.
’ ACKNOWLEDGMENT
This research was supported by the Basic Science Research
Program through the National Research Foundation of Korea
(NRF) funded by the Ministry of Education, Science and Tech-
nology (MEST) (2010-0028112). The work was also funded in
part by a grant from the Brain Korea 21 program and in part by
the NCRCprogramofNRF/MEST (No. 2011-0006244)through
the Center for Cell Signaling & Drug Discovery Research at
Ewha Womans University.
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