Anti-inflammatory triterpenoid saponins from the stem bark of Kalopanax pictus

Article abstract of DOI:10.1021/np200382s

Five new compounds, 16,23,29-trihydroxy-3-oxo-olean-12-en-28-oic acid (1), 4,23,29-trihydroxy-3,4-seco-olean-12-en-3-oate-28-oic acid (2), 3-2-en-28-oic acid 28-O-β-d-glucopyranoside (3), 3-O-[2,3-di-O-acetyl-α-l- arabinopyranosyl]hederagenin 28-O-α-L-rhamnopyranosyl-(→4)-β-d- glucopyranosyl-(→6)-β-d-glucopyranoside (4), and 3-O-[3,4-di-O-acetyl- α-l-arabinopyranosyl]hederagenin 28-O-α-L-rhamnopyranosyl-(→4)- β-d-glucopyranosyl-(→6)-β-d-glucopyranoside (5), as well as 10 known compounds (6-15), were isolated from the stem bark of Kalopanax pictus. Compounds 1-5 and 7-14 inhibited TNFα-induced NF-κB transcriptional activity in HepG2 cells in a dose-dependent manner, with IC₅₀ values ranging from 0.6to 16.4 μM. Furthermore, the transcriptional inhibitory function of these compounds was confirmed on the basis of decreases in COX-2 and iNOS gene expression in HepG2 cells. The structure-activity relationship of the compounds with respect to anti-inflammatory activity is also discussed.

Full text of DOI:10.1021/np200382s

ARTICLE
pubs.acs.org/jnp
Anti-inflammatory Triterpenoid Saponins from the Stem Bark of
Kalopanax pictus
†
,‡
†
‡
‡
†,‡
†
Tran H. Quang, Nguyen T. T. Ngan, Chau V. Minh, Phan V. Kiem, Nguyen X. Nhiem, Bui H. Tai,
†,‡
‡
†
,†
Nguyen P. Thao, Nguyen H. Tung, Seok B. Song, and Young H. Kim*
†
College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea
‡
Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Caugiay, Hanoi,
Vietnam
S Supporting Information
b
ABSTRACT: Five new compounds, 16,23,29-trihydroxy-3-oxo-olean-12-en-28-
oic acid (1), 4,23,29-trihydroxy-3,4-seco-olean-12-en-3-oate-28-oic acid (2),
3
3
β,6β,23-trihydroxyolean-12-en-28-oic acid 28-O-β-D-glucopyranoside (3),
-O-[2,3-di-O-acetyl-α-L-arabinopyranosyl]hederagenin 28-O-α-L-rhamnopyra-
nosyl-(1f4)-β-D-glucopyranosyl-(1f6)-β-D-glucopyranoside (4), and 3-O-
3,4-di-O-acetyl-α-L-arabinopyranosyl]hederagenin 28-O-α-L-rhamnopyrano-
[
syl-(1f4)-β-D-glucopyranosyl-(1f6)-β-D-glucopyranoside (5), as well as 10
known compounds (6ꢀ15), were isolated from the stem bark of Kalopanax
pictus. Compounds 1ꢀ5 and 7ꢀ14 inhibited TNFα-induced NF-kB transcrip-
tional activity in HepG2 cells in a dose-dependent manner, with IC50 values
ranging from 0.6 to 16.4 μM. Furthermore, the transcriptional inhibitory
function of these compounds was confirmed on the basis of decreases in
COX-2 and iNOS gene expression in HepG2 cells. The structureꢀactivity
relationship of the compounds with respect to anti-inflammatory activity is also discussed.
alopanax pictus (Araliaceae) is a deciduous tree growing in
East Asian countries. The stem bark of K. pictus has been
identification of the structures of the known compounds 6ꢀ15
7
K
as nipponogenin E (6), 3β,6β,23-trihydroxyolean-12-en-28-oic
8
3
3
used in traditional medicine to treat rheumatic arthritis, neurotic
acid (7), kalopanaxsaponin A (8), kalopanaxsaponin B (9),
2 9
1
pain, and diabetes mellitus. Phytochemical studies on the stem
kalopanaxsaponin C (10), sieboldianoside A (11), hedera-
10
bark have demonstrated the presence of hederagenin glycosides,
genin 28-O-β-D-glucopyranosyl ester (12), dipsacussaponin
11 12 13
2,3
syringin, liriodendrin, and coniferylaldehyde glucosides, and
A (13), cauloside D (14), and hederagenin (15).
the methanol extract of the stem bark of K. pictus has been
Compound 1 was obtained as a white, amorphous powder.
4
5
reported to possess cytotoxic, antidiabetic, and anti-inflamma-
The molecular formula was determined to be C H O from the
3
0 46 6
6
+
tory activities. In this report, we discuss the isolation and
pseudomolecular ion peak [M + H] at m/z 503.3350 (calcd for
structural elucidation of five new (1ꢀ5) and 10 known com-
pounds (6ꢀ15) from the methanol extract of the stem bark of K.
pictus. The effects of compounds 1ꢀ15 on TNFα (tumor
necrosis factor α)-induced NF-kB transcriptional activity in
HepG2 cells were evaluated. To confirm the inhibitory effects
of the compounds on NF-kB transcriptional activity, we inves-
tigated the effects of the compounds on the upregulation of
the pro-inflammatory proteins iNOS (inducible nitric oxide
synthase) and COX-2 (cyclooxygenase-2) in TNFα-stimulated
HepG2 cells.
C H O , 503.3373) in the HR-ESITOFMS. It showed FT-IR
absorption bands at 3429, 1729, and 1699 cm , suggesting the
30 47 6
ꢀ1
presence of hydroxy, carboxy, and carbonyl groups, respectively.
1
The H NMR spectrum exhibited five quaternary methyl groups
at δ 0.88 (Me-24), 1.02 (Me-25), 0.85 (Me-26), 1.39 (Me-27),
H
and 0.97 (Me-30), a broad singlet for an olefinic proton at δ
5.33 (H-12) characteristic of the Δ proton in pentacyclic
H
12
triterpenes, and a doublet of doublets at δ 3.03 (1H, J =
H
12
14
14.4, 4.2 Hz) attributable to H-18 of the Δ oleanane skeleton.
1
The H NMR spectrum also showed signals for an oxymethine at
δH 4.42 (1H, br s, H-16) and two oxymethylene groups at δH
3.57 (1H, d, J = 11.4 Hz, H-23a), 3.31 (1H, d, J = 11.4 Hz,
H-23b), and 3.16 (2H, d, J = 1.8 Hz, H-29). The C NMR and
DEPT spectra exhibited 30 carbon signals, assignable to five
methyls, 11 methylene, five methine, and nine quaternary
’
RESULTS AND DISCUSSION
13
A MeOH extract of the dried stem bark of K. pictus (290 g) was
suspended in H O and successively extracted with n-hexane,
2
CH Cl , and EtOAc. The EtOAc- and H O-soluble fractions was
2
2
2
subjected to multiple chromatographic steps over Diaion HP-20,
silica gel, and reversed-phase C , yielding compounds 1ꢀ15.
Received: May 3, 2011
Published: August 26, 2011
18
Comparison of the NMR and MS data with reported values led to
Copyright r 2011 American Chemical Society and
American Society of Pharmacognosy
1908
dx.doi.org/10.1021/np200382s |J. Nat. Prod. 2011, 74, 1908–1915

Journal of Natural Products
ARTICLE
Chart 1
1
3
12
carbons. The C NMR spectrum (Table 1) showed signals of a
carbonyl (δC 218.9, C-3), a carboxy (δC 180.2, C-28), two
oxymethylenes [δC 67.1 (C-23) and 73.8 (C-29)], and an
characteristic of the Δ proton in the pentacyclic triterpenes.
1
The H NMR spectrum also showed signals for a methoxy group
at δ 3.59 (3H, s) and oxymethylene protons at δ 3.48 (1H, d,
J = 10.8 Hz, H-23a), 3.18 (1H, d, J = 10.8 Hz, H-23b), and 3.16
(2H, s, H-29). The C NMR and DEPT spectra displayed the
presence of 31 carbons, including six methyls, 11 methylenes, five
methines, and nine quaternary carbons. The C NMR spectrum
(Table 1) of 2 exhibited signals of two carboxy carbons [δ 176.5
(C-3) and 180.8 (C-28)], an oxygenated quaternary carbon (δC
H
H
13
oxymethine carbon (δ 74.0, C-16). The C NMR spectrum
of 1 was similar to that of 23-hydroxy-3-oxolean-12-en-28-oic
acid, except for the presence of an oxymethine and oxymethy-
lene group in 1. The location of a hydroxy group at C-16 was
shown by the HMBC correlation between δ 4.42 (H-16)/δ
C
13
1
5
13
H
C
C
1
1
4
8.9 (C-17) and the Hꢀ H COSY cross-peak between H -15/
2
H-16. The HMBC cross-peaks from δ 3.16 (2H, d, J = 1.8 Hz,
77.1, C-4), and a methoxy carbon (δ 51.1), suggesting that 2
H
C
H-29) to δ 35.7 (C-20) and 19.3 (C-30) permitted placement
of another hydroxy group at C-29. The downfield chemical shifts
possessed a 4-hydroxy-3,4-seco-3-oate ester structural moiety.
This was supported by comparison of the NMR spectra of 2 with
those of the similar compound 3,4-seco-olean-12-en-4-ol-3,28-
C
of C-16 (δ 74.0) and C-29 (δ 73.8) and the broad singlet of
C
C
16
H-16 (δ 4.42) revealed that the C-29 oxymethylene and the
dioic acid. The difference between these two compounds is the
H
C-16 hydroxy group are both α-oriented. On the basis of the
above analyses, the structure of compound 1 was established as
presence of two oxymethylenes and a methoxy group in 2. The
positions of the two oxymethylene groups in 2 at δ 70.6 (C-23)
C
1
6,23,29-trihydroxy-3-oxo-olean-12-en-28-oic acid.
and 73.4 (C-29) were determined by the HMBC cross-peaks
Compound 2 was isolated as a white, amorphous powder, and
(Figure 1) between δ 1.08 (Me-24)/C-23, δ 1.61 (H-5)/C-
H
H
its molecular formula was established as C H O by HR-
23, and Me-30/C-29, respectively. On the basis of these data, the
planar structure of 2 was established. The relative configuration
of 2 was determined by a NOE experiment (Figure 1). In the
NOESY spectrum of 2, the NOE correlations between H-12/H-
11a, H-12/H-18, H-18/30, H -25/H -26, and H -25/H-6a
3
1 50 7
+
ESITOFMS at m/z 535.3651 [M + H] (calcd for C H O ,
31 51 7
5
35.3635). The FT-IR spectrum of 2 revealed hydroxy and
carboxy groups on the basis of absorption bands at 3411 and
ꢀ
1
1
1
701 cm , respectively. The H NMR spectrum of 2 exhibited
the presence of five tertiary methyl singlets at δ 1.08 (Me-24),
3
3
3
revealed the β-orientations of H-11a, H-18, H -30, H -25, H -
H
3
3
3
1
.10 (Me-25), 0.82 (Me-26), 1.15 (Me-27), and 0.90 (Me-30)
26, and H-6a. The NOE cross-peaks between H -27/H-9, H-9/
H-6b, H-6b/H-5, and H-5/H-9 showed the α-orientations of
3
and a broad singlet for an olefinic proton at δH 5.26 (H-12)
1
909
dx.doi.org/10.1021/np200382s |J. Nat. Prod. 2011, 74, 1908–1915

Journal of Natural Products
ARTICLE
H -27, H-9, and H-5. On the basis of these data, the structure of
compound 2 was established as 4,23,29-trihydroxy-3,4-seco-
olean-12-en-3-oat-28-oic acid.
Compounds 3ꢀ5 were isolated as white, amorphous powders.
The monosaccharides obtained after aqueous acid hydrolysis of
each compound were identified as glucose, rhamnose, and
arabinose by TLC comparison with authentic samples. The
absolute configuration of the monosaccharides was determined
to be D for glucose and L for rhamnose and arabinose by GC
analysis of chiral derivatives of the monosaccharides in the
hydrolysate of each compound (see Experimental Section).
The relatively large coupling constants (7.2ꢀ8.4 Hz) for the
3
13
Table 1. C NMR Data for Compounds 1, 2, and Aglycones
a
of Compounds 3ꢀ5
position
1
2
3
4
5
1
anomeric protons in the H NMR spectra (Table 2) of these
1
2
3
4
5
6
7
8
9
37.8
35.7
218.9
47.3
52.5
19.6
32.4
39.6
45.9
36.5
23.6
122.6
144.0
41.9
35.2
74.0
48.9
40.5
41.5
35.7
30.3
30.6
67.1
16.9
14.8
16.7
26.3
180.2
73.8
19.3
34.7
28.9
176.5
77.1
45.2
22.3
32.1
39.4
39.3
41.2
23.2
123.0
143.9
42.5
27.9
23.1
47.0
41.0
40.3
35.9
28.3
32.1
70.6
21.4
19.8
16.9
25.1
180.8
73.4
18.5
51.1
41.8
27.6
73.8
44.3
49.9
68.7
41.2
39.8
49.5
37.5
24.6
124.1
144.3
43.5
29.0
24.1
48.1
42.5
47.3
31.6
35.0
33.2
66.7
14.2
17.8
19.0
26.5
178.2
33.6
24.1
39.5
26.6
82.8
43.9
47.8
18.9
33.4
40.7
49.2
37.7
24.1
123.8
145.0
43.1
29.0
24.7
48.2
42.6
47.3
31.6
35.0
33.4
64.2
13.6
16.6
18.0
26.5
178.2
33.6
24.2
39.5
26.5
83.8
44.0
48.2
18.9
33.3
40.7
49.2
37.7
24.1
123.8
145.0
43.0
29.0
24.6
48.1
42.6
47.3
31.6
35.0
33.4
64.8
13.5
16.7
17.9
26.5
178.2
33.6
24.2
compounds suggested the α-configurations of the arabinopyr-
anosyl moieties and the β-configurations of the glucopyranosyl
moieties. The α-configurations of the rhamnopyranosyl moieties
were determined from the small coupling constants (1.2 Hz)
observed for the anomeric protons (Table 2).
The molecular formula of compound 3 was determined to be
+
C H O from the pseudomolecular ion peak [M + H] at m/z
3
6 58 10
6
51.4133 (calcd for C H O , 651.4108) in the HR-ESI-
36 59 10
TOFMS. Its FT-IR spectrum revealed hydroxy and carboxy
ꢀ
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
0
groups at 3367 and 1731 cm , respectively. The H NMR
1
spectrum exhibited signals for six tertiary methyls at δH 1.02
(
(
Me-24), 1.29 (Me-25), 1.03 (Me-26), 1.10 (Me-27), 0.88
2
Me-29), and 0.90 (Me-30) and a broad singlet for an olefinic
3
12
proton at δ 5.26 (H-12) characteristic of the Δ proton in the
H
4
pentacyclic triterpenes. In addition, an anomeric proton was also
5
1
observed at δH 5.35 (1H, d, J = 8.4 Hz) in the H NMR
6
13
spectrum. The C NMR (Tables 1 and 2) and DEPT spectra
7
exhibited 36 carbon signals, including six methyl, 10 methylene,
8
1
2 methine, and eight quaternary carbons. Comparison of NMR
9
data of 3 with reported data led to identification of the aglycone
of 3 as 3β,6β,23-trihydroxyolean-12-en-28-oic acid. Acid hydro-
8
0
1
lysis of 3 with 10% HCl gave 3β,6β,23-trihydroxyolean-12-en-
28-oic acid and D-glucose. From the above evidence, the structure
of 3 was established to be 3β,6β,23-trihydroxyolean-12-en-28-
oic acid 28-O-β-D-glucopyranoside.
2
3
4
The molecular formula of 4 was determined to be C H O
5
7 90 24
5
+
from the pseudomolecular ion peak [M + H] at m/z 1159.5906
calcd for C H O , 1159.5900) in the HR-ESITOFMS. The
6
(
57 91 24
7
aglycone of 4 was determined as hederagenin by comparison of
8
13
its NMR spectroscopic data with reported values. Acid hydro-
9
lysis of 4 with 10% HCl produced hederagenin, L-arabinose, D-
0
glucose, and L-rhamnose. The anomeric proton signals at δ 4.38
H
-OMe
(1H, d, J = 7.8 Hz), 4.47 (1H, d, J = 7.8 Hz), 4.81 (1H, d, J = 1.2
Hz), and 5.31 (1H, d, J = 8.4 Hz) showed HMQC correlations
a
Spectra were recorded at 150 MHz in methanol-d4.
1
1
Figure 1. Selective Hꢀ H COSY, HMBC, and NOESY correlations of 2.
1
910
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Journal of Natural Products
ARTICLE
1
13
a
Table 2. H (600 MHz) and C NMR (150 MHz) Data for Sugar Moieties of Compounds 3ꢀ5
3
4
5
b
H
position
δ
δ
C
δ
H
δ
C
δ
H
δ
C
3-O-Ara
1
2
3
4
5
4.47, d (7.8)
104.4
74.2
73.0
70.9
64.9
4.44, d (7.2)
3.38
106.3
70.6
74.4
70.0
64.5
4.94
5.01
4.85
3.82
5.17
3.88, dd (12.6, 2.4)
3.87, dd (12.6, 2.4)
3.69, d (12.0)
3.60, d (12.0)
2
3
4
-Ac
2.08, s
21.3
1
72.0
21.1
72.6
-Ac
2.11, s
1.99, s
2.07, s
20.9
172.3
20.9
1
-Ac
172.2
2
1
2
3
4
5
6
8-O-Glc
5.35, d (8.4)
3.29
95.9
74.0
78.3
71.2
78.7
62.5
5.31, d (8.4)
3.30
95.9
73.8
78.2
70.7
76.8
69.5
5.32, d (8.4)
3.31
95.8
73.8
78.2
70.9
76.7
69.4
3.39
3.50
3.39
3.32
3.94, dd (9.6, 6.0)
3.43, t (9.6)
3.77
3.60
3.32
3.43
3.79
3.76
3
.65, dd (12.0, 4.2)
4.06, d (11.4)
4.07, d (10.2)
Glc
1
4.38, d (7.8)
3.20, t (9.0)
3.26
104.3
75.4
76.9
79.6
78.2
61.9
4.38, d (7.8)
3.21, t (8.4)
3.27
104.3
75.3
76.8
79.5
78.1
61.9
2
3
4
3.52
3.51
5
3.38
3.38
6
3.62
3.63
3.78
3.77
Rha
1
4.81, d (1.2)
3.80
103.0
72.5
72.3
73.9
71.0
17.9
4.82, d (1.2)
3.81
102.9
72.4
72.2
73.8
70.7
18.0
2
3
3.61
3.61
4
3.38
3.38
5
3.37
3.37
6
1.24, d (6.6)
1.24, d (6.0)
a
b
Spectra were recorded in methanol-d
4
. Coupling constants (J) are in Hz. Protons are overlapped, unless otherwise stated (s, singlet; d, doublet; t, triplet).
with anomeric carbon signals at δ 104.3, 104.4, 103.0, and 95.9,
respectively, indicating that 4 possessed four sugar units. The
C-28-glucose (δ 69.5), and H-1 of C-28-glucose (δ 5.31) with
C H
C
C-28 of the aglycone (δ 178.2). In addition to the aglycone and
C
downfield chemical shift of C-3 (δC 82.8) and the upfield
sugar chains, two additional acetyl signals were observed in the
NMR spectra. Attachment of the two acetyl groups to C-2 and
C-3 of the arabinose was confirmed from HMBC correlations
between H-2 (δ 4.94) and H-3 (δ 5.01) of arabinose with the
13
chemical shift of C-28 (δ 178.2) in the C NMR spectrum
C
of 4 (Table 1) indicated that this compound is a bisdesmosidic
1
13
saponin. The H and C NMR data (Table 2) of the mono-
saccaride residues and the sequence of the sugar residues of 4
were assigned unambiguously on the basis of the HMQC and
HMBC spectra. The location of the arabinose at C-3 was
identified from the HMBC cross-peak between H-1 of arabinose
C
C
carbonyl carbons of the two acetyl groups (δ 172.0 and 172.6),
C
respectively. On the basis of the data obtained, the structure of 4
was identified as 3-O-[2,3-di-O-acetyl-α-L-arabinopyranosyl]-
hederagenin 28-O-α-L-rhamnopyranosyl-(1f4)-β-D-glucopyranosyl-
(1f6)-β-D-glucopyranoside.
(
δ 4.47) and C-3 of the aglycone (δ 82.8). The sugar chain at
H C
+
C-28 was also established as α-L-rhamnopyranosyl-(1f4)-β-D-
glucopyranosyl-(1f6)-β-D-glucopyranosyl from the following
HMBC correlations: H-1 of rhamnose (δH 4.81) with C-4
of glucose (δ 79.6), H-1 of glucose (δ 4.38) with C-6 of
The HR-ESITOFMS of 5 exhibited the ion peak [M + H] at
m/z 1159.5876 (calcd for C H O , 1159.5900), consistent
57 91 24
with the molecular formula C H O . Acid hydrolysis of 5 with
57
90 24
10% HCl gave hederagenin, L-arabinose, D-glucose, and L-rhamnose.
C
H
1
911
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Journal of Natural Products
ARTICLE
Figure 2. Effects of compounds 1ꢀ15 on the TNFα-induced NF-kB luciferase reporter activity in HepG2 cells. The values are mean ( SD (n = 6).
a
b
Stimulated with TNFα. Stimulated with TNFα in the presence of 1ꢀ15 (0.1, 1, and 10 μM) and sulfasalazine. SFZ: sulfasalazine, positive control
(
10 μM). Statistical significance is indicated as * (p < 0.05) or ** (p < 0.01) as determined by Dunnett’s multiple comparison test.
The downfield chemical shift of C-3 (δ 83.8) and the upfield
Table 3. Inhibitory Effects of Compounds 1ꢀ15 on the
C
1
3
a
chemical shift of C-28 (δ 178.2) in the C NMR spectrum of 5
TNFα-Induced NF-kB Transcriptional Activity
C
(
Table 1) revealed that 5 is a bisdesmosidic saponin. Linkage of the
compound
IC50 (μM)
compound
IC50 (μM)
sugar units was determined by the HMQC and HMBC experi-
ments. Comparison of the NMR spectroscopic data of 5 with those
of 4 (Tables 1 and 2) revealed that these compounds possessed the
same trisaccharide moiety linked to C-28 of the aglycone. The
1
2
3
4
5
6
7
8
6.5 ( 0.35
9.4 ( 0.55
16.4 ( 0.54
8.9 ( 0.65
9.3 ( 0.75
9
0.8 ( 0.05
3.9 ( 0.19
0.6 ( 0.03
9.1 ( 0.63
6.2 ( 0.25
9.2 ( 0.54
>20
10
11
12
13
14
15
HMBC correlation of H-1 of the arabinose [δ 4.44 (1H, d, J = 7.2
H
Hz)] with C-3 of the aglycone (δC 83.8) indicated that the
arabinose was located at C-3. Moreover, two acetyl groups linked
to C-3 and C-4 of the arabinose were identified from HMBC
correlations between H-3 (δ 4.85) and H-4 (δ 5.17) of arabinose
and the carbonyl carbons of the two acetyl groups (δ 172.3 and
1
of 5 was established as 3-O-[3,4-di-O-acetyl-α-L-arabinopyrano-
syl]hederagenin 28-O-α-L-rhamnopyranosyl-(1f4)-β-D-glucopyra-
nosyl-(1f6)-β-D-glucopyranoside.
The anti-inflammatory activity of compounds 1ꢀ15 was
evaluated through inhibition of a TNFα-induced NF-kB lucifer-
ase reporter and by attenuation of TNFα-induced pro-inflam-
matory gene (iNOS and COX-2) expression in HepG2 cells.
The NF-kB luciferase assay is designed to monitor the activity
of NF-kB-regulated signal transduction pathways in cultured
cells. The NF-kB-responsive luciferase construct encodes the
firefly luciferase reporter gene under the control of a minimal
CMV promoter and tandem repeats of the NF-kB transcriptional
response element. Using this assay, the inhibitory activity of
compounds 1ꢀ15 on NF-kB activation is readily monitored.
Pro-inflammatory agents, such as TNFα, are known to activate
b
>20
1.4 ( 0.27
5.5 ( 0.32
H
H
sulfasalazine
b
0.9 ( 0.2
C
a
The values are mean ( SD (n = 6). A compound is considered
inactive with IC50 > 20 μM.
72.2), respectively. On the basis of this information, the structure
ranging from 0.6 to 16.4 μM (Table 3). Among the compounds
tested, compounds 7, 9, and 11 showed the most potent effects, with
IC50 values of 1.4, 0.8, and 0.6 μM, respectively. Remarkably, the
inhibitory effects of compounds 9and 11 were more potent than that
of the anti-inflammatory chemotherapy drug sulfasalazine (IC50
.9 μM). Other compounds (1ꢀ5, 8, 10, and 12ꢀ14) exhibited
significant activity with IC values ranging from 3.9 to 16.4 μM.
=
0
50
Since NF-kB is an important transcription factor involved in
regulating the expression of inflammatory NF-kB target genes such
18,19
as iNOS and COX-2,
we investigated the effects of compounds
1
ꢀ5 and 7ꢀ14 on the expression of these genes in TNFα-
stimulated HepG2 cells using RT-PCR. HepG2 cells treated with
0 ng/mL TNFα significantly upregulated the mRNA expression of
1
17
the NF-kB pathway. HepG2 cells transfected with the NF-kB
luciferase reporter plasmid exhibited approximately a 9-fold
increase in the luciferase signal after treatment with 10 ng/mL of
TNFα, indicating that transcriptional activity increased compared to
untreated cells. The results showed that compounds 1ꢀ5 and 7ꢀ14
inhibited TNFα-induced NF-kB transcriptional activity in HepG2
cells in a dose-dependent manner (Figure 2), with IC50 values
the NF-kB target genes COX-2 and iNOS. Consistent with their
inhibitory activity toward NF-kB, compounds 1ꢀ5 and 7ꢀ14
significantly inhibited the induction of COX-2 and iNOS mRNA in
a dose-dependent manner (Figure 3), indicating that these com-
pounds reduced transcription of these genes. Moreover, the house-
keeping protein β-actin was not changed by the presence of
compounds 1ꢀ5 and 7ꢀ14 at the same concentration.
1
912
dx.doi.org/10.1021/np200382s |J. Nat. Prod. 2011, 74, 1908–1915

Journal of Natural Products
ARTICLE
injection temperature, 250 °C; injection volume, 0.5 μL. HR-ESITOF
mass spectra were obtained using a JEOL JMS-T100LC spectrometer.
The NMR spectra were recorded on a JEOL ECA 600 spectrometer
using TMS as an internal standard. TLC was performed on Kieselgel 60
F254 (1.05715; Merck, Darmstadt, Germany) or RP-18 F254s (Merck)
plates. Spots were visualized by spraying with 10% aqueous H SO4
2
solution, followed by heating. CC was performed on silica gel (Kieselgel
6
0, 70ꢀ230 mesh and 230ꢀ400 mesh, Merck) and YMC RP-18 resins.
Plant Material. The stem bark of of K. pictus was purchased from an
herbal market at Kumsan, Chungnam, Korea, in August 2009. The plant
material was identified by one of us (Y.H.K.). A voucher specimen
(CNU09105) was deposited at the herbarium, College of Pharmacy,
Chungnam National University.
Extraction and Isolation. The dried stem bark of K. pictus (3 kg)
was extracted with hot MeOH. After concentration, the MeOH extract
(290 g) was suspended in H
2
O and then partitioned successively with n-
hexane, CH Cl , and EtOAc to give n-hexane (A, 50 g), CH Cl (B,
2
2
2
2
30 g), EtOAc (C, 15 g), and water (D, 195 g) fractions, respectively.
Fraction C was chromatographed over silica gel, eluting with MeOH in
CH Cl (0ꢀ100%, stepwise), yielding five fractions (C1ꢀC5). Fraction
2
2
Figure 3. Effects of compounds 1ꢀ5 and 7ꢀ14 on iNOS and COX-2
C1 (2 g) was chromatographed over a silica gel column eluting with
mRNA expression in HepG2 cells.
CH
2
Cl
2
ꢀMeOH (10:1) and further purified by YMC RP chromatog-
O (3:1) as eluent to afford 1 (6 mg) and 2
10 mg). Fraction C2 (2.2 g) was chromatographed over a silica gel
raphy using MeOHꢀH
2
The anti-inflammatory activity, in combination with the
structural properties of compounds 1ꢀ15, allowed us to infer
certain guidelines regarding the structureꢀactivity relationship.
The results showed that compound 15 was not active, whereas all
saponins were active, suggesting that the sugar moieties play
essential roles in the anti-inflammatory activity. The hydroxy
group at C-6 also plays an important role in the anti-inflamma-
tory activity on the basis of a comparison of the structure and
activity of compound 15 with those of 3 and 7. The structures of
compounds 4, 5, 9, 10, 11, and 14 were found to be similar,
except for the sugar moieties located at C-3 of the aglycones.
When the arabinose unit at C-3 of the aglycones linked to other
saccharides, such as a rhamnose (9), a rhamnose-xylose chain
(
column eluting with CH
2
Cl
2
ꢀMeOH (17:1) to obtain 6 (10 mg) and 7
(16 mg). Fraction C4 (1.7 g) was separated by YMC RP chromatog-
raphy using MeOHꢀH O (3:1) as eluent to give 12 (45 mg) and 15 (20
2
mg). Fraction D was chromatographed on a column of highly porous
polymer (Diaion HP-20) and eluted with H
to give three fractions (D1ꢀD3). Fraction D2 (26 g) was chromato-
graphed over silica gel, eluting with MeOH in CH Cl
(0ꢀ100%,
2
O and MeOH, successively,
2
2
stepwise), to provide five subfractions (D2AꢀD2E). Subfraction D2C
(
(
CH
2.3 g) was separated by YMC RP chromatography, using MeOHꢀH
1:1) as eluent, and further purified by CC over silica gel, eluting with
Cl O (5:1:0.1), to obtain 9 (55 mg) and 11 (65 mg).
2
O
2
2
ꢀMeOHꢀH
2
Subfraction D2D (3.5 g) was separated by CC over silica gel, using
(
11), or a rhamnose and glucose (10), the anti-inflammatory
CH Cl ꢀMeOHꢀH O (4:1:0.1) as eluents, and further purified by
2
2
2
activity increased significantly as compared to single arabinose
units at C-3 of the aglycones of 4, 5, and 14. Also, the acetyl
groups on the arabinose units in compounds 4 and 5 did not
affect the activity compared to the activity of compounds 4, 5,
and 14. Moreover, the carbonyl (C-3) and hydroxy group at C-16
of compound 1 and the ring A-3,4-seco of 2 also played important
roles in the anti-inflammatory activity, on the basis of comparison
of the structure and activity of compounds 1, 2, and 6. These data
may be useful in evaluating the structureꢀfunction relationship
of other oleanane-type saponins and their aglycons, as well as to
develop novel anti-inflammatory agents for medical uses.
YMC RP chromatography, eluting with MeOHꢀH
2
O (1:1), to give 10
57 mg). Fraction D3 (35 g) was chromatographed over silica gel,
(
eluting with MeOH in CH Cl (0ꢀ100%, stepwise), to provide four
2
2
subfractions (D3AꢀD3D). Subfraction D3B (6.8 g) was then separated
by CC over silica gel, using CH Cl ꢀMeOHꢀH O (5:1:0.1) as eluents,
2
2
2
and further purified by YMC RP chromatography, eluting with
MeOHꢀH O (2:1), to afford 3 (16 mg), 8 (18 mg), and 13 (15 mg).
2
Subfraction D3D (5.6 g) was separated by YMC RP chromatography,
using MeOHꢀH
over silica gel, eluting with CH
4 (10 mg), 5 (9 mg), and 14 (23 mg).
2
O (15:10) as eluent, and further purified by CC
Cl O (4:1:0.1), to obtain
ꢀMeOHꢀ H
2
2
2
1
6,23,29-Trihydroxy-3-oxo-olean-12-en-28-oic acid (1): white, amor-
Cell viability, as measured by the MTT [3-(4,5-dimethylthia-
2
5
phous powder; [α]
429, 2931, 1729, 1699, 1456, 1376, 1260, 1035, 862, 799 cm ; H NMR
D 3
+4.8 (c 0.09, MeOH); FT-IR (CH CN) νmax
zol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric
ꢀ1 1
2
0
3
method, showed that compounds 1ꢀ5 and 7ꢀ14 had no
significant cytotoxicity in HepG2 cells at concentrations that
effectively inhibited NF-kB activation and induction of COX-2
and iNOS gene expression (data not shown).
4
data (methanol-d , 600 MHz) δ 5.33 (1H, br s, H-12), 4.42 (1H, br s,
H-16), 3.57 (1H, d, J = 11.4 Hz, H-23a), 3.31 (1H, d, J = 11.4 Hz, H-23b),
.16 (2H, d, J = 1.8 Hz, H-29), 3.03 (1H, dd, J = 14.4, 4.2 Hz, H-18), 1.85
1H, m, H-15a), 1.39 (3H, s, Me-27), 1.37 (1H, m, H-15b), 1.02 (3H, s,
Me-25), 0.97 (3H, s, Me-30), 0.88 (3H, s, Me-24), 0.85 (3H, s, Me-26);
3
(
’
EXPERIMENTAL SECTION
1
3
C NMR data (methanol-d , 150 MHz), see Table 1; HR-ESITOFMS
4
+
General Experimental Procedures. Optical rotations were
m/z 503.3350 [M + H] (calcd for C30
47 6
H O , 503.3373).
determined using a Jasco DIP-370 digital polarimeter. The FT-IR
spectra were measured using a Jasco Report-100 infrared spectrometer.
ESI mass spectra were obtained using an Agilent 1200 LC-MSD Trap
spectrometer. GC was carried out on a Shimadzu-2010 spectrometer:
detector, FID; detection temperature, 300 °C; column, SPB-1 (0.25 mm
i.d. ꢁ 30 m); column temperature, 230 °C; carrier gas, He (2 mL/min);
4 23,29-Trihydroxy-3,4-seco-olean-12-en-3-oat-28-oic acid (2): white,
25
amorphous powder; [α]
3411, 2935, 1701, 1456, 1436, 1384, 1265, 1199, 1175, 1042, 933 cm ; H
NMR data (methanol-d , 600 MHz) δ 3.59 (3H, s, MeO-3), 1.15 (3H, s,
+40 (c 0.04, MeOH); FT-IR (CH CN) νmax
D
3
ꢀ
1 1
4
Me-27), 1.10 (3H, s, Me-25), 1.08 (3H, s, Me-24), 0.90 (3H, s, Me-30),
0.82 (3H, s, Me-26), 5.26 (1H, br s, H-12), 3.48 (1H, d, J = 10.8 Hz,
1
913
dx.doi.org/10.1021/np200382s |J. Nat. Prod. 2011, 74, 1908–1915

Journal of Natural Products
ARTICLE
2
+
H-23a), 3.18 (1H, d, J = 10.8 Hz, H-23b), 3.16 (2H, s, H-29), 2.87 (1H, dd,
J = 13.8, 4.2 Hz, H-18), 2.59 (1H, m, H-2a), 2.39 (1H, m, H-1a), 2.13 (1H,
m, H-2b), 1.98 (1H, m, H-16a), 1.87 (1H, m, H-11a), 1.80 (1H, m, H-9),
Mg and an excess of ATP. Under these conditions, luciferase enzymes
expressed by the reporter vector could catalyze the oxidative carboxyla-
5
tion of luciferin. Cells were seeded at 1.5 ꢁ 10 cells per well in 12-well
1
.77 (1H, m, H-19a), 1.75 (1H, m, H-7a), 1.71 (1H, m, H-15a), 1.61 (1H,
m, H-5), 1.57 (3H, m, H-1b, H-11b, H-16b), 1.56 (1H, m, H-22a), 1.47
1H, m, H-6a), 1.46 (1H, m, H-7b), 1.45 (1H, m, H-21a), 1.28 (1H, m,
H-6b), 1.23 (1H, m, H-22b), 1.10 (1H, m, H-21b), 1.07 (1H, m, H-15b),
plates and grown for 24 h. All cells were transfected using WelFect M
Gold (WelGENE Inc., Daegu, South Korea), as guided by the manu-
facturer. Luciferase activity was assayed using an LB 953 Autolumat
(
2
1
(EG&G Berthold, Nashua, NH). The transfected HepG2 cells were
pretreated for 1 h with either vehicle (DMSO) or compounds, followed
by 1 h of treatment with 10 ng/mL TNFα. Unstimulated HepG2 cells
were used as a negative control (ꢀ). Cells were then harvested, and
luciferase activity was assayed. The NF-kB-luciferase plasmid was kindly
provided by Dr. Kyoon E. Kim (Chungnam National University,
Daejeon, Korea).
13
1
.05 (1H, m, H-19b); C NMR data (methanol-d
4
, 150 MHz), see
Table 1; HR-ESITOFMS m/z 535.3651 [M + H] (calcd for C31
35.3635).
,6β,23-Trihydroxyolean-12-en-28-oic acid 28-O-β-D-glucopyrano-
+
51 7
H O ,
5
3
25
side (3): white, amorphous powder; [α]
D
+55.3 (c 0.1, MeOH); FT-IR
(
9
CH CN) ν
3367, 2928, 1731, 1456, 1384, 1231, 1063, 1028,
3 max
ꢀ
1 1
33 cm ; H NMR data (methanol-d , 600 MHz) δ 1.29 (3H, s,
RNA Preparation and Reverse Transcriptase Polymerase
Chain Reaction (RT-PCR). Total RNA was extracted using Easy-blue
reagent (Intron Biotechnology, Seoul, Korea). Approximately 2 μg of
total RNA was subjected to reverse transcription using Moloney murine
leukemia virus reverse transcriptase and oligo-dT primers (Promega,
Madison, WI) for 1 h at 42 °C. PCR for synthetic cDNA was performed
using a Taq polymerase premixture (TaKaRa, Japan). The PCR
products were separated by electrophoresis on 1% agarose gels and
stained with EtBr. PCR was conducted with the following primer pairs:
4
Me-25), 1.10 (3H, s, Me-27), 1.03 (3H, s, Me-26), 1.02 (3H, s, Me-24),
.90 (3H, s, Me-30), 0.88 (3H, s, Me-29), 5.26 (1H, br s, H-12), 4.34
1H, br s, H-6), 3.57 (1H, d, J = 10.8 Hz, H-23a), 3.53 (1H, dd, J = 12.0,
0
(
4
13
.2 Hz, H-3), 3.44 (1H, d, J = 10.8 Hz, H-23b); C NMR data
(
methanol-d
4
, 150 MHz), see Tables 1 and 2; HR-ESITOFMS m/z
+
6
51.4133 [M + H] (calcd for C36
-O-[2,3-Di-O-acetyl-α-L-arabinopyranosyl]hederagenin 28-O-α-L-
rhamnopyranosyl-(1f4)-β-D-glucopyranosyl-(1f6)-β-D-glucopyra-
59
H O10, 651.4108).
3
25
0 0
iNOS sense 5 -TCATCCGCTATGCTGGCTAC-3 , iNOS antisense
5 -CTCAGGGTCACGGCCATTG-3 , COX-2 sense 5 -GCCCAG-
noside (4): white, amorphous powder; [α]
IR (CH CN) ν 3367, 2929, 1732, 1434, 1371, 1237, 1023 cm ; H
D
+11 (c 0.1, MeOH); FT-
ꢀ
1
1
0
0
0
3
max
0
0
NMR data (methanol-d , 600 MHz) δ 1.13 (3H, s, Me-27), 0.94 (3H, s,
CACTTCACGCATCAG-3 , COX-2 antisense 5 -GACCAGGCAC-
4
0 0
CAGACCAAAGACC-3 , β-actin sense 5 -TCACCCACACTGTGC-
Me-25), 0.91 (3H, s, Me-30), 0.88 (3H, s, Me-29), 0.76 (3H, s, Me-26),
0
0
0
.55 (3H, s, Me-24), 5.22 (1H, br s, H-12), 3.81 (1H, d, J = 11.4 Hz,
CCATCTACG-3 , and β-actin antisense 5 -CAGCGGAACCGCT-
CATTGCCAATG-3 . The specificity of products generated by each
1
3
0
H-23a), 3.60 (1H, m, H-3), 3.29 (1H, d, J = 11.4 Hz, H-23b); C NMR
data (methanol-d , 150 MHz), see Tables 1 and 2; HR-ESITOFMS m/z
159.5906 [M + H] (cacld for C57
-O-[3,4-Di-O-acetyl-α-L-arabinopyranosyl]hederagenin 28-O-α-L-
rhamnopyranosyl-(1f4)-β-D-glucopyranosyl-(1f6)-β-D-glucopyra-
set of primers was examined using gel electrophoresis and further
confirmed by a melting curve analysis.
HepG2 cells were pretreated in the absence and presence of
compounds for 1 h, then exposed to 10 ng/mL TNFα for 6 h. Total
mRNA was prepared from the cell pellets using Easy-blue. The levels of
mRNA were assessed by RT-PCR.
4
+
1
91
H O24, 1159.5900).
3
25
noside (5): white, amorphous powder; [α]
FT-IR (CH CN) νmax 3368, 2929, 1727, 1448, 1373, 1228, 1029 cm
H NMR data (methanol-d , 600 MHz) δ 1.14 (3H, s, Me-27), 0.96
D
+6.3 (c 0.17, MeOH);
ꢀ
1
3
;
1
4
(
3H, s, Me-25), 0.91 (3H, s, Me-30), 0.88 (3H, s, Me-29), 0.77 (3H, s,
’
ASSOCIATED CONTENT
Me-26), 0.69 (3H, s, Me-24), 5.22 (1H, br s, H-12), 3.58 (1H, d, J = 11.4
Hz, H-23a), 3.60 (1H, m, H-3), 3.26 (1H, d, J = 11.4 Hz, H-23b);
NMR data (methanol-d , 150 MHz), see Tables 1 and 2; HR-ESI-
91
TOFMS m/z 1159.5876 [M + H] (cacld for C57H O24, 1159.5900).
1
3
C
S
b
Supporting Information. FT-IR, 1D and 2D NMR, and
4
HR-ESI-TOF mass spectra for the new compounds 1ꢀ5. This
material is available free of charge via the Internet at http://pubs.
acs.org.
+
Acid Hydrolysis of Saponins. Each saponin (4 mg) was heated in
mL of 10% HClꢀdioxane (1:1) at 80 °C for 3 h. After the solvent was
3
2
removed in vacuo, and the residue was partitioned between EtOAc and H O
’
AUTHOR INFORMATION
to give the aglycone and the sugar, respectively. The sugar components in the
aqueous layer were analyzed by silica gel TLC by comparison with standard
sugars. The solvent system was CH Cl ꢀMeOHꢀH O (2:1:0.2), and
Corresponding Author
*Tel: 82-42-821-5933. E-mail: yhk@cnu.ac.kr.
2
2
2
spots were visualized by spraying with 95% EtOHꢀH
2
SO
4
ꢀanisaldehyde
(9:0.5:0.5, v/v), then heated at 180 °C for 5 min. The R
f
values of glucose,
arabinose, and rhamnose byTLC was 0.30, 0.5, and 0.75, respectively. The
results were confirmed by GC analysis. The aqueous layer was evaporated
todryness togive a residue, dissolved inanhydrous pyridine(100 μL), and
then mixed with a pyridine solution of 0.1 M L-cysteine methyl ester
hydrochloride (100 μL). After warming at 60 °C for 2 h, trimethylsily-
limidazole solution was added, and the reaction solution was warmed at
’ ACKNOWLEDGMENT
This work was supported by a grant from the Korea Food and
Drug Administration for Studies on Standardization of Herbal
Medicine (2010) and Priority Research Center Program through
the National Research Foundation of Korea (NRF) funded by
the Ministry of Education, Science and Technology (2009-
6
0 °C for 2 h. The mixture was evaporatedin vacuo to give a dried product,
0
093815), Republic of Korea.
which was partitioned between n-hexane and H O. The n-hexane layer
2
was filtered and analyzed by GC. The retention times of the persilylated
monosaccharide derivatives were as follows: L-arabinose (t , 4.72), L-
’ REFERENCES
R
R R
rhamnose (t , 5.31 min), and D-glucose (t , 14.11 min) were confirmed
by comparison with those of authentic standards.
In Vitro Anti-inflammatory Assay. NF-jB-Luciferase Assay.
The NF-kB-luciferase plasmid was first transfected into HepG2 cells.
After a limited amount of time, the cells were lysed and luciferin, the
substrate of luciferase, was introduced into the cellular extract along with
(
1) Kim, T. J. Korean Resources Plants; Seoul National University
Press: Seoul, 1996; Vol. 2, pp 169ꢀ200.
2) Shao, C.-J.; Kasai, R.; Xu, J.-D.; Tanaka, O. Chem. Pharm. Bull.
(
1989, 37, 311–314.
(3) Sano, K.; Sanada, S.; Ida, Y.; Shoji, J. Chem. Pharm. Bull. 1991,
39, 865–870.
1
914
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Journal of Natural Products
ARTICLE
(
4) Lee, K. T.; Sohn, I. C.; Park, H. J.; Kim, D. W.; Jung, K. O.; Park,
K. Y. Planta Med. 2000, 66, 329–332.
5) Park, H. J.; Kim, D. H.; Choi, J. W.; Park, J. H.; Han, Y. N. Arch.
Pharm. Res. 1998, 21, 24–29.
6) Lee, E. B.; Li, D. W.; Hyun, J. E.; Kim, I. H.; Whang, W. K.
J. Ethnopharmacol. 2001, 77, 197–201.
7) Miyakoshi, M.; Shirasuna, K.; Hirai, Y.; Shingu, K.; Isoda, S.;
Shoji, J.; Ida, Y.; Shimizu, T. J. Nat. Prod. 1999, 62, 445–448.
8) Khan, I. A.; Sticher, O.; Rali, T. J. Nat. Prod. 1993,
6, 2163–2165.
9) Sawada, H.; Miyakoshi, M.; Isoda, S.; Ida, Y.; Shoji, J. Phyto-
chemistry 1993, 34, 1117–1121.
10) Panov, D. A.; Grishkovets, V. I.; Kachala, V. V.; Shashkov, A. S.
Chem. Nat. Compd. 2006, 42, 61–66.
11) Kizu, H.; Hirabayashi, S.; Suzuki, M.; Tomimori, T. Chem.
Pharm. Bull. 1985, 33, 3473–3478.
12) Strigina, L. I.; Chetyrina, N. S.; Isakov, V. V.; Elkin, Y. N.;
Dzizenko, A. K.; Elyakov, G. B. Phytochemistry 1975, 14, 1583–1586.
13) Tori, K.; Sera, S.; Shimaoka, A.; Tomita, Y. Tetrahedron Lett.
974, 15, 4227–4230.
14) Shamma, M.; Glick, R. E.; Mumma, R. O. J. Org. Chem. 1962,
7, 4512–4517.
15) Verma, D. K.; Singh, S. K.; Nath, G. Indian Drugs 1997,
4, 390–392.
16) Shirane, N.; Hashimoto, Y.; Ueda, K.; Takenaka, H.; Katoh, K.
Phytochemistry 1996, 43, 99–104.
(
(
(
(
5
(
(
(
(
(
1
2
3
(
(
(
(17) Paul, A. G. Eukaryon 2005, 1, 4–5.
(18) Wong, H. R.; Menendez, I. Y. Biochem. Biophys. Res. Commun.
1
999, 262, 375–380.
(19) Pahl, H. L. Oncogene 1999, 18, 6853–6866.
(20) Scudiero, D. A.; Shoemaker, R. H.; Paull, K. D.; Monks, A.;
Tierney, S.; Nofziger, T. H.; Currens, M. J.; Seniff, D.; Boyd, M. R.
Cancer Res. 1988, 48, 4827–4833.
(21) Kim, K. K.; Park, K. S.; Song, S. B.; Kim, K. E. Mol. Carcinog.
2
010, 49, 259–270.
1
915
dx.doi.org/10.1021/np200382s |J. Nat. Prod. 2011, 74, 1908–1915