Cytotoxic triterpene saponins from the stem bark of Kalopanax pictus

Article abstract of DOI:10.1016/j.phytol.2011.12.005

Three new compounds, 3β,6β,23-trihydroxyolean-12-en-28-oic acid 3-O-α-l-arabinopyranoside (1), kalopanaxsaponin L (2), and kalopanaxsaponin M (13), as well as eleven known compounds (3-12 and 14), were isolated from the stem bark of Kalopanax pictus. Their structures were determined on the basis of extentive spectroscopic analyses and acid hydrolysis. The cytotoxicity of the compounds was evaluated in three human carcinoma cell lines, including HL-60, HCT-116, and MCF-7. Compounds 1, 5-8, 10, and 11 exhibited significantly cytotoxic activity toward HL-60 cells, with IC ₅₀ values ranging from 0.1 to 6.9 μM. Compounds 4-7 and 14 showed significant cytotoxicity against HCT-116 cells, with IC₅₀ values ranging from 0.4 to 9.2 μM. Remarkably, the cytotoxic activities of compounds 5-7 against HCT-116 cells were greater than that of the anticancer chemotherapy drug, mitoxantrone (IC₅₀ = 3.7 μM). Compounds 1, 3, 5, and 14 were cytotoxic toward MCF-7 cells with IC₅₀ values in a range of 7.4-14.5 μM.

Full text of DOI:10.1016/j.phytol.2011.12.005

Phytochemistry Letters 5 (2012) 177–182
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Cytotoxic triterpene saponins from the stem bark of Kalopanax pictus
Tran Hong Quang a,b, Nguyen Thi Thanh Ngan a, Chau Van Minh b, Phan Van Kiem b,
Hye-Jin Boo c, Jin-Won Hyun c, Hee-Kyoung Kang c, Young Ho Kim a,
*
a College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea
b Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Caugiay, Hanoi, Viet Nam
c School of Medicine, Institute of Medical Sciences, Jeju National University, Jeju 690-756, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history:
Three new compounds, 3b,6b,23-trihydroxyolean-12-en-28-oic acid 3-O-a-L-arabinopyranoside (1),
Received 17 August 2011
kalopanaxsaponin L (2), and kalopanaxsaponin M (13), as well as eleven known compounds (3–12 and
14), were isolated from the stem bark of Kalopanax pictus. Their structures were determined on the basis
of extentive spectroscopic analyses and acid hydrolysis. The cytotoxicity of the compounds was
evaluated in three human carcinoma cell lines, including HL-60, HCT-116, and MCF-7. Compounds 1, 5–
8, 10, and 11 exhibited significantly cytotoxic activity toward HL-60 cells, with IC50 values ranging from
Received in revised form 5 December 2011
Accepted 8 December 2011
Available online 20 December 2011
Keywords:
0.1 to 6.9
values ranging from 0.4 to 9.2
116 cells were greater than that of the anticancer chemotherapy drug, mitoxantrone (IC50 = 3.7
Compounds 1, 3, 5, and 14 were cytotoxic toward MCF-7 cells with IC50 values in a range of 7.4–14.5
m
M. Compounds 4–7 and 14 showed significant cytotoxicity against HCT-116 cells, with IC50
M. Remarkably, the cytotoxic activities of compounds 5–7 against HCT-
M).
M.
Kalopanax pictus
Araliaceae
m
m
m
Triterpene saponin
Cytotoxic
ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
1. Introduction
multiple chromatographic steps over Diaion HP-20, silica gel, and
reversed-phase C18, yielding compounds 1–14. Comparison of the
The stem bark of Kalopanax pictus (Araliaceae), a deciduous tree
that grows in East Asian countries, has been used in traditional
medicine to treat rheumatic arthritis, neurotic pain, and diabetes
mellitus (Kim, 1996). Phytochemical studies on the stem bark have
demonstrated the presence of hederagenin glycosides, syringin,
liriodendrin, and coniferylaldehyde glucosides (Sano et al., 1991;
Shao et al., 1990) and a methanol extract of the stem bark of K.
pictus has been reported to possess cytotoxic (Lee et al., 2000),
antidiabetic (Park et al., 1998), and anti-inflammatory activities
(Lee et al., 2001). In this report, we discuss the isolation and
structural elucidation of three new compounds (1, 2, and 13), along
with eleven known compounds (3–12 and 14), from a methanol
extract of the stem bark of K. pictus. The cytotoxicity of the
compounds was evaluated in three human carcinoma cell lines:
leukemia (HL-60), colon (HCT-116), and breast (MCF-7).
NMR and MS data with reported values led to the identification of
the structures of known compounds: 3 ,6 ,23-trihydroxyolean-
12-en-28-oic acid (3) (Khan et al., 1993), hederagenin (4) (Tori
et al., 1974), hederagenin 3-O- -L-arabinopyranoside (5) (Shimizu
et al., 1978), kalopanaxsaponin A (6) (Sano et al., 1991), sapindo-
side B (7) (Sawada et al., 1993), hederagenin 28-O- -glucopyr-
anosyl ester (8) (Panov et al., 2006), cussonoside A (9) (Dubois
et al., 1986), cauloside D (10) (Strigina et al., 1975), cussonoside B
(11) (Dubois et al., 1986), tarasaponin IV (12) (Satoh et al., 1994),
and caulophyllogenin (14) (Strigina et al., 1974) (see Fig. 1).
Compounds 1, 2, and 13 were isolated as white amorphous
powders. The relatively large coupling constants (6.6–8.4 Hz) for
the anomeric protons in the 1H NMR spectra of these compounds
b
b
a
b-D
suggested the
and the -configurations of the glucopyranosyl, glucuronopyr-
anosyl, and galactopyranosyl moieties. The -configurations of the
a-configurations of the arabinopyranosyl moieties
b
a
rhamnopyranosyl and arabinofuranosyl moieties were determined
from broad singlet signals of the anomeric protons.
2. Results and discussion
The molecular formula of compound 1 was determined to be
35H56O9 from a pseudomolecular ion peak at m/z 621.4032
[M+H]+ (calcd for C35H57O9, 621.4003) in the HR-ESI-TOF MS. It
showed FT-IR absorption bands at 3369 and 1653 cmꢀ1, suggesting
the presence of hydroxyl and carbonyl groups, respectively. The 1H
A MeOH extract of the dried stem bark of K. pictus (290 g) was
suspended in H2O and successively extracted with n-hexane,
CH2Cl2, and EtOAc. The H2O-soluble fraction was subjected to
C
NMR spectrum showed six tertiary methyl groups at
dH 1.04 (Me-
* Corresponding author. Tel.: +82 42 821 5933; fax: +82 42 823 6566.
E-mail address: yhk@cnu.ac.kr (Y.H. Kim).
24), 1.30 (Me-25), 1.10 (Me-26), 1.09 (Me-27), 0.85 (Me-29), and
1874-3900/$ – see front matter ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.phytol.2011.12.005

178
T.H. Quang et al. / Phytochemistry Letters 5 (2012) 177–182
Table 1
HMQC, and HMBC spectra obtained for this compound, and
13C NMR (150 MHz) data for compounds 1, 2, and 13.
comparison with those in the literature (Lacaille-Dubois et al.,
1997; Melek et al., 2004). The linkage of the sugar units at C-3 of
the aglycone was established from the HMBC correlations between
Position
1a
2b
13a
40.0
1
2
41.8
38.2
H-1 of galactose (
between H-1 of glucuronic acid (dH 4.29) and C-3 of the aglycone
dC 78.7) (Fig. 2). Thus, the sugar chain attached to C-3 was
determined to be 3-O- -glucur-
-galactopyranosyl-(1 ! 2)-
onopyranosyl. Similarly, the sugar chain at C-28 was also
established to be -D-glucopyra-
-L-rhamnopyranosyl-(1 ! 4)-
nosyl-(1 ! 6)- -D-glucopyranosyl from the following HMBC
dH 4.28) and C-2 of glucuronic acid (dC 82.5), and
26.5
83.5
44.9
50.0
68.7
41.4
39.7
49.6
37.3
24.6
123.0
145.8
43.7
29.3
24.6
48.7
43.5
48.1
31.9
35.6
34.4
64.8
15.1
17.9
19.3
26.6
185.2
34.0
24.4
24.9
78.7
42.3
46.0
17.2
31.7
38.2
47.2
35.9
23.0
121.8
143.5
41.3
27.3
22.5
46.0
40.7
45.5
30.3
33.3
31.9
62.3
12.8
15.7
16.7
25.5
175.4
32.8
23.4
27.2
91.4
40.5
57.1
19.5
34.1
40.8
49.0
38.0
24.7
124.0
144.9
43.0
29.0
24.1
48.2
42.6
47.3
31.6
35.0
33.4
28.6
17.1
16.2
18.0
26.4
178.2
33.6
24.2
3
4
(
5
b
-D
b-D
6
7
a
b
8
9
b
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
correlations: H-1 of rhamnose (dH 4.64) with C-4 of glucose (dC
76.4), and H-1 of glucose (dH 4.23) with C-6 of C-28-glucose (dC
67.5), and H-1 of C-28-glucose (
C 175.4) (Fig. 2). On the basis of the above analysis, the structure
of 2 was identified as 3-O- -D-
-D-galactopyranosyl(1 ! 2)-
glucuronopyranosyl hederagenin 28-O- -L-rhamnopyranosyl-
(1 ! 4)- -D-glucopyranosyl-(1 ! 6)- -D-glucopyranoside,
named kalopanaxsaponin L (see Fig. 1).
dH 5.16) with C-28 of the aglycone
(
d
b
b
a
b
b
The HR-ESI-TOF MS of 13 exhibited an ion peak [M+H]+ at m/z
1413.6545 (calcd for C65H105O33, 1413.6538), consistent with the
molecular formula C65H104O33. Acid hydrolysis of 5 with 10% HCl
gave oleanolic acid, L-arabinose, D-glucose, and D-glucuronic acid.
The anomeric proton signals at dH 4.32 (1H, d, J = 8.4 Hz), 4.34 (1H,
d, J = 7.2 Hz), 4.42 (1H, d, J = 7.2 Hz), 4.67 (1H, d, J = 7.8 Hz), 5.04
(1H, br s), and 5.34 (1H, d, J = 7.8 Hz) showed HMQC cross-peaks
with anomeric carbon signals at dC 104.9, 105.0, 105.7, 104.7,
109.6, and 95.8, respectively, indicating that 13 possessed six sugar
units. The downfield chemical shift of C-3 (dC 91.4) and the upfield
a
Spectra were recorded in CD3OD.
chemical shift of C-28 of the aglycone (dC 178.2) in the 13C NMR
spectra of 13 (Table 1) indicated this compound to be bisdesmo-
sidic saponin. The 1H and 13C NMR data (Tables 2 and 3) of the
monosaccharides and the sequence of the sugar residues of 13
were assigned unambiguously on the basis of the HMQC and HMBC
spectra, and comparison with those in the literature (Miyase et al.,
b
DMSO-d6.
0.92 (Me-30), a broad singlet for an olefinic proton at
12) characteristic of the D12 proton in the pentacyclic triterpenes,
d
H 5.23 (H-
and a doublet doublet at
to H-18 of D12 oleanane skeleton (Shamma et al., 1962). In
addition, an anomeric proton was also observed at H 4.30 (1H, d,
J = 6.6 Hz) in the 1H NMR spectrum. The 13C NMR (Tables 1 and 3)
and DEPT spectra exhibited 35 carbon signals, including 6 methyl,
10 methylene, 11 methine, and 8 quaternary carbons. Comparison
of the NMR data of 1 with those in the literature led to the
d
H 2.89 (1H, J = 13.8, 4.2 Hz) attributable
1996). The sugar chain at C-3 was established to be 3-O-
arabinofuranosyl(1 ! 4)-[ -D-glucopyranosyl(1 ! 2)]- -D-glu-
curonopyranosyl from the HMBC cross-peaks as follows: H-1 of
arabinose ( H 5.04) with C-4 of C-3-glucuronic acid ( C 79.5), H-1 of
glucose ( H 4.67) with C-2 of C-3-glucuronic acid ( C 81.0), and H-1
of C-3-glucuronic acid ( H 4.42) with C-3 of the aglycone ( C 91.4)
(Fig. 2). In the same way, the sugar chain at C-28 was identified as
28-O-
a-L-
b
b
d
d
d
d
d
d
d
identification of the aglycone of 1 as 3
en-28-oic acid (Khan et al., 1993). Acid hydrolysis of 1 with 10% HCl
afforded 3 ,6 ,23-trihydroxyolean-12-en-28-oic acid and L-arab-
inose. Thus, the structure of 1 was established as 3 ,6 ,23-
-L-arabinopyranoside
b
,6b
,23-trihydroxyolean-12-
b
-D-glucopyranosyl(1 ! 6)-
b
-D-glucopyranosyl(1!6)-
b-
D-glucopyranosyl based on the following HMBC correlations: H-1
of glucose (dH 4.34) with C-6 of glucose (dC 69.9), H-1 of glucose
b
b
b
b
(
dH 4.32) with C-6 of C-28-glucose (dC 69.9), and H-1 of C-28-
glucose (dH 5.34) with C-28 of the aglycone (dC 178.2) (Fig. 2).
Thus, the structure of 13 was established as 3-O- -L-ara-
binofuranosyl(1 ! 4)-[ -D-glucopyranosyl(1 ! 2)]- -D-glucuro-
nopyranosyl oleanolic acid 28-O- -D-
-D-glucopyranosyl(1 ! 6)-
-D-glucopyranoside, named kalopanax-
trihydroxyolean-12-en-28-oic acid 3-O-
a
(see Fig. 1).
a
The molecular formula of 2 was determined as C60H96O29 by
HR-ESI-TOF MS, which showed a pseudomolecular ion peak at m/z
1281.6063 [M+H]+ (calcd for C60H97O29, 1281.6116). The aglycone
of 2 was identified as hederagenin by comparison of its NMR
spectroscopic data with reported values (Tori et al., 1974). Acid
hydrolysis of 2 with 10% HCl gave hederagenin and D-glucose, D-
glucuronic acid, D-galactose, and L-rhamnose. The anomeric
proton signals at dH 4.23 (1H, d, J = 7.8 Hz), 4.28 (1H, d,
J = 7.2 Hz), 4.29 (1H, d, J = 7.2 Hz), 4.64 (1H, s), and 5.16 (1H, d,
J = 7.8 Hz) gave correlations with anomeric carbon signals at dC
102.5, 105.1, 101.9, 100.5, and 94.0 in the HMQC spectrum,
respectively, indicating that 2 contained five sugar units. The
b
b
b
b
glucopyranosyl(1 ! 6)-
b
saponin M (see Fig. 1).
The cytotoxicity of compounds 1–14 against HL-60, HCT-116,
and MCF-7 cells was evaluated using the MTT assay (Scudiero et al.,
1988). The results (Table 4) showed that compounds 1 and 7
exhibited the most potent cytotoxicity against HL-60 cells, with
IC50 values of 0.3 and 0.1
11, and 14 displayed significant cytotoxicity toward HL-60 cells,
with IC50 values ranging from 4.6 to 13.8 M, while compounds 3
and 4 exhibited moderate cytotoxicity against HL-60, with IC50
values of 26.4 and 25.1 M, respectively. Compounds 5–7 showed
the best cytotoxic activity toward HCT-116 cells, with IC50 values
of 0.5, 0.4, and 0.8 M, respectively. Remarkably, the cytotoxic
mM, respectively. Compounds 5, 6, 8, 10,
m
downfield chemical shift of C-3 (
d
C 78.7) and the upfield chemical
m
shift of C-28 of the aglycone (
d
C 175.4) in the 13C NMR spectra of 2
(Table 1) indicated this compound to be bisdesmosidic saponin.
The 1H and 13C NMR data (Tables 2 and 3) of the glucuronic acid,
galactose, glucose, and rhamnose units were assigned starting
from the readily identifiable anomeric protons by means of the
m
activities of compounds 5–7 against HCT-116 cells were greater
than that of the anticancer chemotherapy drug, mitoxantrone
(Table 4). Compounds 1, 3, 5, and 14 displayed significant

T.H. Quang et al. / Phytochemistry Letters 5 (2012) 177–182
179
Table 2
1H NMR (600 MHz) data for sugar moieties of compounds 1, 2, and 13.
Position
1a,*
2b,*
13a,*
Position
2b,*
13a,*
3-O-Ara(p)
28-O-Glc
1
2
3
4
5
4.30 (d, 6.6)
1
2
3
4
5
5.16 (d, 7.8)
5.34 (d, 7.8)
3.53 (dd, 9.0, 6.6)
3.47 (dd, 9.0, 3.6)
3.76
3.16 (dd, 8.4, 7.8)
3.30 (dd, 9.0, 8.4)
3.12 (dd, 9.0, 8.4)
3.21
3.31 (dd, 8.4, 7.8)
3.62 (t, 9.0)
3.33 (dd, 9.0, 8.4)
3.32
3.81 (dd, 12.0, 3.0)
3.51 (br d, 12.0)
6
3.86 (br d, 11.4)
3.56 (br d, 11.4)
4.14 (dd, 11.4, 1.8)
3.75 (br d, 11.4)
3-O-GlcA
Glc
1
1
2
3
4
5
6
4.29 (d, 7.2)
4.42 (d, 7.2)
4.23 (d, 7.8)
3.21
4.32 (d, 8.4)
3.17 (dd, 9.0, 7.2)
3.01 (t, 9.0)
3.64 (dd, 9.0, 7.2)
3.20 (dd, 9.6, 9.0)
3.69 (dd, 9.6, 9.0)
3.17 (d, 9.0)
2
3.19 (dd, 9.0, 8.4)
3.61 (dd, 9.6, 9.0)
3.22 (dd, 9.0, 8.4)
3.18
3
3.14 (t, 9.0)
3.32 (t, 9.0)
3.18
3.15 (dd, 9.0, 8.4)
3.41 (d, 8.4)
4
5
6
3.36 (br d, 12.0)
3.55 (br d, 12.0)
4.06 (brd, 12.0)
3.71 (br d, 12.0)
Gal
1
Glc
1
4.28 (d, 7.2)
4.34 (d, 7.2)
2
3.31
2
3.19 (dd, 9.0, 7.2)
3.24 (t, 9.0)
3
3.23 (dd, 8.4, 7.8)
3
4
3.62
2.93
3.48
3.31
4
3.23 (t, 9.0)
5
5
3.78
6
6
3.85 (dd, 11.4, 2.4)
3.65 (dd, 11.4, 3.6)
Glc
1
Rha
1
4.67 (d, 7.8)
4.64 (br s)
2
3.64 (dd, 9.0, 7.8)
3.40 (dd, 9.6, 8.4)
3.39 (dd, 9.0, 8.4)
3.32
2
3.56
3
3
3.35 (dd, 9.0, 2.4)
3.07 (t, 9.0)
3.81 (dd, 9.0, 6.0)
1.04 (d, 6.0)
4
4
5
5
6
3.79 (br d, 12.0)
3.68 (dd, 12.0, 3.0)
6
Ara(f)
1
2
3
4
5
5.04 (br s)
3.95 (dd, 3.6, 1.8)
3.19
4.07 (dd, 9.0, 4.8)
3.79 (dd, 12.0, 4.8)
3.58 (11.4, 3.6)
a
Spectra were recorded in CD3OD.
b
in DMSO-d6. Coupling constants (J) are in Hz.
*
Protons are overlapped, unless otherwise stated (s, singlet; d, doublet; t, triplet).
cytotoxicity toward MCF-7 cells, with IC50 values ranging from 7.4
to 14.5 M (Table 4).
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 H2SO4 solution, followed
by heating. Column chromatography (CC) was performed on silica
gel (Kieselgel 60, 70–230 mesh and 230–400 mesh, Merck) and
YMC RP-18 resins.
m
From the results, we found that hederagenin (4) and its
monodesmosides (5–8) showed more effective and broader
cytotoxic activity than its bidesmosides (only compound 10 was
cytotoxic toward HL-60 cells). Furthermore, hederagenin 3-O-
glycosides (1 and 5–7) were found to show broader cytotoxicity
activity than hederagenin 28-O-glycosides (8 and 11). Compound 3
and its 3-O-glycoside (1) were cytotoxic against the three cell lines
tested. Oleanolic acid monodesmoside (11) exhibited cytotoxicity
against HL-60 cells, whereas its bidesmosides (12 and 13) showed
no activity. These data suggest that the free carboxyl group (C-28)
plays an important role in cytotoxic activity of the oleanane-type
triterpene saponins.
3.2. Plant materials
The stem bark of K. pictus was purchased from herbal market at
Kumsan, Chungnam, Korea, in August 2009. The plant material was
identified by one of us (Y.H. Kim). A voucher specimen (CNU09105)
was deposited at herbarium, College of Pharmacy, Chungnam
National University.
3. Experimental
3.3. Extraction and isolation
3.1. General experimental procedures
The dried stem bark of K. pictus (3 kg) was extracted with hot
MeOH. After concentration, the MeOH extract (290 g) was
suspended in water and then partitioned successively with
n-hexane, CH2Cl2, and EtOAc to give n-hexane (A), CH2Cl2 (B),
EtOAc (C) and water (D) fractions, respectively. Fraction C was
chromatographed over silica gel, eluting with MeOH in CH2Cl2
(0–100%, step-wise), yielding five fractions (C1–C5). Fraction C2
Optical rotation was determined using a Jasco DIP-370 digital
polarimeter. The FT-IR spectra were measured using a Jasco
Report-100 infrared spectrometer. Electrospray ionization (ESI)
mass spectra were obtained using an Agilent 1200 LC-MSD Trap
spectrometer. HR-ESI-TOF mass spectra were obtained using a
JEOL JMS-T100LC spectrometer. The NMR spectra were recorded

180
T.H. Quang et al. / Phytochemistry Letters 5 (2012) 177–182
Table 3
13C NMR (150 MHz) data for sugar moieties of compounds 1, 2, and 13.
Position
1a
2b
13a
Position
2b
13a
3-O-Ara(p)
28-O-Glc
1
106.2
73.1
74.6
69.8
66.8
1
94.0
95.8
2
2
72.2
77.0
69.3
75.0
67.5
73.9
78.0
71.5
78.0
69.9
3
3
4
4
5
5
3-O-GlcA
6
1
101.9
82.5
75.5
71.8
76.7
173.1
105.7
81.0
Glc
1
2
102.5
73.4
75.2
76.4
76.5
59.9
104.9
75.2
78.1
71.6
78.3
69.9
3
76.5
79.5
77.2
176.3
2
4
3
5
4
6
5
Gal
1
6
105.1
72.6
73.0
67.3
73.8
59.2
Glc
1
2
105.0
75.2
77.8
71.9
78.7
62.8
3
2
4
3
5
4
6
5
6
Glc
Rha
1
1
104.7
77.0
78.2
71.0
78.0
63.1
100.5
70.7
70.6
72.0
68.6
17.8
2
2
3
3
4
4
5
5
6
6
Ara(f)
1
2
3
4
5
109.6
83.0
75.2
86.4
63.3
a
Spectra were recorded in CD3OD.
b
DMSO-d6.
was chromatographed over silica gel column eluting with CH2Cl2–
MeOH (17:1) to obtain 3 (15 mg) and 14 (16 mg). Fraction C4 was
separated by YMC reverse-phase chromatography using MeOH–
H2O (3:1) as eluents to give 4 (20 mg) and 8 (55 mg). Fraction C5
was chromatographed over silica gel column eluting with CH2Cl2–
MeOH (10:1), and further purified by YMC reverse-phase
chromatography using MeOH–H2O (2:1) as eluents to afford 1
(7 mg), 7 (10 mg), and 5 (6 mg). Fraction D was chromatographed
on a column of highly porous polymer (Diaion HP-20) and eluted
with H2O and MeOH, successively to give three fractions (D1–D3).
Fraction D2 was chromatographed over silica gel, eluting with
MeOH in CH2Cl2 (0–100%, step-wise) to provide five subfractions
Fig. 1. Structures of compounds 1–14.
Table 4
Cytotoxicity data for compounds 1–14.
(D2A–D2E). Subfraction D2B was separated by YMC reverse-phase
chromatography, using MeOH–H2O (1:1) as eluents, and further
purified by column chromatography over silica gel, eluting with
CH2Cl2–MeOH–H2O (3:1:0.1) to obtain 12 (15 mg) and 13 (20 mg).
Subfraction D2C was separated by column chromatography over
silica gel, using CH2Cl2–MeOH–H2O (4:1:0.1) as eluents, and
further purified by YMC reverse-phase chromatography, eluting
with MeOH–H2O (1:1) to give 2 (25 mg). Fraction D3 was
chromatographed over silica gel, eluting with MeOH in CH2Cl2
(0–100%, step-wise) to provide four subfractions (D3A–D3D).
Subfraction D3B was then separated by column chromatography
over silica gel, using CH2Cl2–MeOH–H2O (5:1:0.1) as eluents, and
further purified by YMC reverse-phase chromatography, eluting
with MeOH–H2O (2:1) to afford 6 (18 mg). Subfraction D3D
was separated by YMC reverse-phase chromatography, using
MeOH–H2O (15:10) as eluents, and further purified by column
Compound
Cell lines IC50
(mM)
HL-60
HCT-116
MCF-7
1
0.3 ꢁ 0.08
26.4 ꢁ 0.4
25.1 ꢁ 1.5
5.8 ꢁ 0.1
5.6 ꢁ 0.1
0.1 ꢁ 0.03
4.6 ꢁ 0.3
6.9 ꢁ 0.3
5.1 ꢁ 0.7
13.8 ꢁ 1.2
0.1 ꢁ 0.01
25.0 ꢁ 0.2
18.4 ꢁ 1.0
9.2 ꢁ 0.1
0.5 ꢁ 0.03
0.4 ꢁ 0.06
0.8 ꢁ 0.07
>30a
13.8 ꢁ 1.4
11.8 ꢁ 1.5
>30
3
4
5
7.4 ꢁ 0.8
>30
6
7
>30
8
>30
10
>30
>30
11
>30
>30
14
6.3 ꢁ 0.1
3.7 ꢁ 0.6
14.5 ꢁ 2.3
7.1 ꢁ 0.5
Mitoxantroneb
The values are mean ꢁ SD (n = 3).
Compounds 2, 9, 12, and 13 were inactive against all cell lines.
a
A compound is considered inactive with IC50 > 30 mM.
Positive control.
b

T.H. Quang et al. / Phytochemistry Letters 5 (2012) 177–182
181
O
O
O
OH
HOOC
O
HOOC
HO
OH
O
O
HO
OH
O
O
HO
O
O
O
HO
O
OH
OH
O
OH
OH
OH
OH
HOH
C
O
O
O
HO
OH
O
O
O
O
HO
OH
HO
OH
1
HO
OH
O
O
HO
HO
O
HO
HO
OH
OH
O
O
HO
HO
OH
HO
HO
H
HO
C
O
O
OH
HO
OH
HO
HO
HO
2
13
Fig. 2. Selective HMBC correlations of compounds 1, 2, and 13.
chromatography over silica gel, eluting with CH2Cl2–MeOH–H2O
(4:1:0.1) to 9 (35 mg), 10 (23 mg) and 11 (12 mg).
samples, in MeCOEt–iso-PrOH–Me2CO–H2O (20:10:7:6). After
separation by preparative TLC of the sugar mixture, the optical
rotation of each purified monosaccharide was measured and
identified as D-glucose, D-galactose, L-rhamnose, L-arabinose, and
D-glucuronic acid (Voutquenne et al., 2005).
3.4. 3b,6b,23-trihydroxyolean-12-en-28-oic acid 3-O-a-L-
arabinopyranoside (1)
A white, amorphous powder; [
a
]
25 +21.3 (c 0.1, MeOH); FT-IR
3.8. Cytotoxic assay
D
(CH3CN)
n
max 3369, 2925, 1653, 1541, 1449, 1374, 1046 cmꢀ1
.
1H
NMR data (DMSO-d6, 600 MHz)
d
H 5.23 (1H, br s, H-12), 4.39 (1H,
Human cancer cell lines including leukemia (HL-60), colon
(HCT-116), and breast (MCF-7) were purchased from the Korea Cell
Line Bank (KCLB, Seoul, Korea). Cytotoxic assay was performed
using the MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetra-
zolium bromide] colorimetric method (Scudiero et al., 1988).
br s, H-6), 3.65 (1H, d, J = 10.8 Hz, H-23a), 3.46 (1H, d, J = 10.8 Hz,
H-23b), 2.89 (1H, dd, J = 13.8, 4.2 Hz, H-18), 1.30 (3H, s, Me-25),
1.10 (3H, s, Me-26), 1.09 (3H, s, Me-27), 1.04 (3H, s, Me-24), 0.92
(3H, s, Me-30), 0.85 (3H, s, Me-29); 13C NMR data (DMSO-d6,
150 MHz), see Tables 1 and 3; HR-ESI-TOF MS: m/z 621.4032
[M+H]+ (calcd for C35H57O9, 621.4003).
Acknowledgements
3.5. Kalopanaxsaponin L (2)
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 (2009-0093815),
Republic of Korea.
25
A white, amorphous powder; [
FT-IR (CH3CN) nmax 3166, 2861, 1731, 1614, 1428, 1031 cmꢀ1
NMR data (DMSO-d6, 600 MHz)
m, H-3), 3.47 (1H, d, J = 10.8 Hz, H-23a), 2.93 (1H, d, J = 10.8 Hz, H-
23b), 1.03 (3H, s, Me-27), 0.82 (6H, s, Me-25, Me-29), 0.80 (3H, s,
Me-30), 0.62 (3H, s, Me-26), 0.51 (3H, s, Me-24); 13C NMR data
(DMSO-d6, 150 MHz), see Tables 1 and 3; HR-ESI-TOF MS: m/z
1281.6063 [M+H]+ (cacld for C60H97O29, 1281.6116).
a
]
ꢀ15.4 (c 0.19, MeOH 50%);
1H
H 5.12 (1H, br s, H-12), 3.48 (1H,
D
.
d
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