Triterpene saponins from the aerial parts of Ilex cornuta and their cytotoxic activity

Article abstract of DOI:10.3987/COM-15-13175

Four new triterpene saponins (1-4), together with 13 known triterpenoids (5-17), were isolated from the MeOH extract of Ilex cornuta Lindley (Aquifoliaceae). Their structures were elucidated on the basis of chemical and spectroscopic methods. The isolated compounds were tested for their cytotoxicities against four human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and HCT15) in vitro using the sulforhodamine B (SRB) assay.

Full text of DOI:10.3987/COM-15-13175

HETEROCYCLES, Vol. 91, No. 6, 2015
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HETEROCYCLES, Vol. 91, No. 6, 2015, pp. 1187 - 1197. © 2015 The Japan Institute of Heterocyclic Chemistry
Received, 17th January, 2015, Accepted, 15th April, 2015, Published online, 11th May, 2015
DOI: 10.3987/COM-15-13175
TRITERPENE SAPONINS FROM THE AERIAL PARTS OF ILEX
CORNUTA AND THEIR CYTOTOXIC ACTIVITY
Seung Young Lee,a Won Se Suh,a Ho Kyung Kim,a Il Kyun Lee,b Sang
Un Choi,c Ki Hyun Kim,a and Kang Ro Lee*,a
aNatural Products Laboratory, School of Pharmacy, Sungkyunkwan University;
Suwon 440-746, Korea. bNatural Products Research Team, Richwood
Pharmaceutical Company Ltd, 694-1 Sampyeong-dong, Bundang-gu, Seongnam,
c
Gyeonggi-do 463-400, Republic of Korea. Korea Research Institute of Chemical
Technology, Deajeon 305-600, Korea, E-mail : krlee@skku.edu
Abstract - Four new triterpene saponins (1-4), together with 13 known
triterpenoids (5-17), were isolated from the MeOH extract of Ilex cornuta Lindley
(Aquifoliaceae). Their structures were elucidated on the basis of chemical and
spectroscopic methods. The isolated compounds were tested for their
cytotoxicities against four human tumor cell lines (A549, SK-OV-3, SK-MEL-2,
and HCT15) in vitro using the sulforhodamine B (SRB) assay.
Ilex cornuta Lindley (the Chinese holly or horned holly) belongs to the family Aquifoliaceae, and this
herb is native to eastern China and Korea.1 This species have been used in traditional Chinese medicine
for the treatment of intertrigo, headache, dizziness, and hypertension, and for purifying the blood.2 The
water extract from its leaves has been used as contraceptive and anti-bacterial agents.3 Previous
phytochemical studies on this plant reported the isolation of various triterpene saponins as main
constituents such as ilexside, zigu-glucoside, cornutaside, and cornutaoside.3-7 Some of these isolates
have been reported to exhibit increase in coronary blood flow and anti-hematoblastic coagulation
activity.5
We have recently reported the isolation of triterpene derivatives from the MeOH extract of I. cornuta.8 In
continuing research on this source, four new triterpene saponins (1-4), together with 13 known
triterpenoids (5-17) were isolated and identified (Figure. 1). The structures were determined using

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HETEROCYCLES, Vol. 91, No. 6, 2015
spectroscopic methods including 1D and 2D NMR (1H, 13CꢀNMR, COSY, HMQC, HMBC, and ROESY)
and HR-MS data as well as chemical methods. Herein, we report the isolation, structural elucidation of
the new compounds, and cytotoxic effects of these isolated compounds (1-17).
30
29
20
HO
19
O
OH
26
28
22
R2
19
11
O
O
28
OR3
17
11
H
26
25
H
25
17
1
20
14
27
21
1
2
29
14
2
H
30
27
R1O
R1O
H
R2
H
23
24
23
24
R1
Ara
R2
H, OH
O
R1
Ara
R2
OH
H
R3
H
Glc
3
1
4 Glc-(1→2)-Ara
2 2'-acetoxy-Ara
Figure 1. Structures of Compounds 1-4
Compound 1, a colorless gum, has the molecular formula C35H56O9 as determined by the positive ion
HRFAB-MS m/z 643.3825 [M + Na]+ (calcd. for C35H56NaO9 643.3822). The 13C NMR and DEPT
spectrum (Table 1) exhibited 35 carbon signals composed of one carboxylic carbon at δC 180.8, six
methyl carbons at δC 29.9, 24.5, 17.4, 16.2, 16.1, and 13.7, two olefinic carbons at δC 139.6, and 127.3,
one oxygenated methine carbons at δC 82.1, one oxygenated methylene carbon at δC 64.5, four methine
carbons at δC 48.0, 47.7, 47.5, and 42.2, nine methylene carbons at δC 38.9, 33.3, 32.5, 29.3, 27.1, 26.2,
25.0, 24.1 and 18.4, six quaternary carbons at δC 73.2, 47.9, 43.6, 42.2 40.3 and 37.0, and five remaining
signals at δC 106.7, 74.6, 73.5, 69.7, and 67.0 assignable to arabinose moiety. The 1H NMR spectrum of 1
(Table 2) displayed the signals of an olefinic proton at δH 5.57 (1H, br t, H-12), one oxygenated methine
proton at δH 4.26 (1H, m, H-3), a couple of oxymethylene protons at δH 4.28 and 3.70 (each 1H, d, J =
11.2 Hz, H-23), one methine proton at δH 3.29 (1H, s, H-18), five tertiary methyl protons at δH 1.70 (3H, s,
H-27), 1.44 (3H, s, H-29), 1.11 (3H, s, H-26), 0.99 (3H, s, H-25) and 0.94 (3H, s, H-24), one secondary
methyl proton at δH 1.14 (3H, d, J = 6.0 Hz, H-30), and one sugar anomeric protons at δH 4.99 (1H, d, J =
7.0 Hz, H-1′). The NMR spectral data were very similar to those of mateside (10),9 except for
stereochemistry of methyl group at C-30.10 The β-orientation of the methyl group at C-30 was determined
from correlations of the methyl group CH3-30 (GH 1.14)/CH3-29 (GH 1.44) and H-18 (GH 3.29) in the
ROESY spectrum (Figure. 2). And, upfield shift of C-18 (GC 47.5 in 1; GC 54.7 in 10) and C-22 (GC 32.5 in
1; GC 38.6 in 10) can be explained by γ-effect for the 30β-methyl group.11,12 The above data suggested that

HETEROCYCLES, Vol. 91, No. 6, 2015
1189
the absolute configuration of C-20 was to be 20S.11,12 Acid hydrolysis of compound 1 with HCl gave
aglycone, 20(S)-rotundic acid, and L-arabinose, which were identified by GC analysis with authentic
samples. Therefore, compound 1 was identified as 3β,19α,23-trihydroxy-20(S)-urs-12-en-28-oic acid
3β-O-α-L-arabinopyranoside.
Table 1. 13C NMR Data of Compounds 1-4 (pyridine –d5, 125 MHz, δ in ppm)
Carbon
1
1
2
3
4
38.9 t
26.2 t
82.1 d
43.6 s
47.7 d
18.4 t
33.3 t
40.3 s
48.0 d
37.0 s
24.1 t
127.3 d
139.6 s
42.2 s
29.3 t
27.1 t
47.9 s
47.5 d
73.2 s
43.1 d
25.0 t
32.5 t
64.5 t
13.7 q
16.1 q
17.4 q
24.5 q
180.8 s
29.9 s
16.2 d
106.7 d
73.5 d
38.8 t
26.9 t
88.9 d
39.3 s
55.3 d
18.8 t
33.7 t
40.6 s
47.9 d
37.1 s
24.1 t
127.8 d
138.8 s
42.3 s
29.2 t
26.9 t
48.5 s
47.3 d
73.6 s
42.9 d
24.8 t
32.0 t
28.1 q
17.6 q
15.6 q
16.9 q
24.4 q
177.1 s
29.8 s
16.1 d
104.4 d
72.6 d
40.0 t
27.1 t
88.8 d
39.5 s
56.4 d
18.7 t
35.5 t
41.5 s
50.2 d
37.7 s
33.4 t
69.6 d
150.9 s
43.7 s
28.0 t
29.9 t
46.1 s
120.4 d
68.6 d
46.0 d
78.1 d
43.7 t
28.6 q
17.0 q
17.2 q
19.3 q
22.6 q
179.7 s
21.9 q
10.6 q
107.9 d
73.3 d
39.6 t
26.5 t
88.7 d
39.0 s
56.0 d
18.3 t
35.0 t
41.2 s
49.7 d
37.2 s
32.9 t
69.1 d
150.8 s
43.3 s
27.5 t
29.3 t
45.3 s
119.5 d
209.0 s
51.6 d
77.3 d
43.3 t
28.2 q
16.6 q
16.7 q
18.8 q
22.2 q
178.7 s
29.3 q
13.1 q
104.8 d
81.0 d
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1'
2'

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HETEROCYCLES, Vol. 91, No. 6, 2015
3'
74.6 d
69.7 d
67.0 t
74.4 d
69.7 d
67.1 t
95.9 d
74.2 d
79.3 d
71.3 d
79.0 d
62.4 t
170.2
21.3
75.0 d
69.9 d
67.2 t
73.4 d
68.3 d
65.0 t
106.0 d
76.4 d
78.1 d
71.6 d
78.2 d
62.5 t
4'
5'
1''
2''
3''
4''
5''
6''
2'-OAc
Compound 2 was obtained as a colorless gum. Its molecular formula C43H68O14 was inferred from the
positive ion HRESI-MS m/z 831.4500 [M + Na]+ (calcd. for C43H68NaO14 831.4507). The NMR spectra
of 2 were very similar to those of brevicuspisaponin 3 (13).13 The major differences were the presence of
additional acetyl group signals (GH 2.05, GC 170.2 and 21.3).5 The position of acetyl group was confirmed
by HMBC experiment, which showed correlations between the H-2' (GH 5.88) of α-arabinose and a
carbonyl carbon (GC 170.2).5 The stereochemistry of 2 was established based on the ROESY correlation
between CH3-30/CH3-29 and H-18, suggested that the methyl group at C-30 was of β-orientation (Figure
2). This demonstrated the axial position of methyl group at C-30, indicating the 20S-configuration.11,12
Acid hydrolysis of 2 with 1N HCl gave two sugars, D-glucose and L-arabinose, which were identified by
GC analysis and TLC comparison with authentic samples.7,14 Thus, the structure of 2 was established as
3-O-α-L-2'-acetoxyl-arabinopyranosyl-3β,19α-dihydroxy-20(S)-urs-12-en-28-oic acid 28-O-β-D-glucopy-
ranoside.
Table 2. 1H NMR Data of Compounds 1-4 (pyridine –d5, 500 MHz, δ in ppm)
Position
1
2
3
4
3.38 dd
3.25 dd
H-3
4.26 m
3.18 m
(11.5, 4.5)
(11.5, 4.5)
H-12
H-18
H-19
H-20
H-21
5.57 br t
3.29 s
5.45 br t
2.71 s
4.63 m
6.16 br s
4.02 m
1.78 m
5.08 m
4.58 m
6.16 br s
2.78 m
4.82 m
2.11 m
2.28 m
2.08 m
2.24 m
H-22

HETEROCYCLES, Vol. 91, No. 6, 2015
1191
4.28 d (11.0)
H-23
3.70 d (11.0)
CH3-23
CH3-24
CH3-25
CH3-26
CH3-27
CH3-29
CH3-30
H-1'
1.13 s
1.29 s
1.25 s
0.94 s
0.94 s
0.94 s
1.02 s
0.99 s
0.85 s
0.83 s
0.83 s
1.11 s
1.12 s
1.30 s
1.27 s
1.70 s
1.67 s
1.25 s
1.24 s
1.44 s
1.34 s
1.34 d (7.0)
1.02 d (7.0)
4.72 d (7.0)
2.16 s
1.14 d (6.0)
4.99 d (7.0)
0.94 d (6.0)
4.64 d (7.0)
1.20 d (7.0)
4.99 d (5.5)
5.88 dd
H-2'
(9.5, 7.0)
H-1''
OAc
6.28 d (8.0)
2.05 s
5.19 d (7.5)
Compound 3 was obtained as a colorless gum whose molecular formula was determined to be C35H56O9
1
from the [M + Na]+ peak at m/z 643.3820 (calcd. for C35H56NaO9 643.3822) in the HRESI-MS. The H
NMR spectrum of 3 displayed signals for seven methyl groups at GH 0.83 (3H, s, H-25), 0.94 (3H, s,
H-24), 1.02 (3H, d, J = 7.0 Hz, H-30), 1.25 (3H, s, H-27), 1.29 (3H, s, H-23), 1.30 (3H, s, H-26), and 1.34
(3H, d, J = 7.0 Hz, H-29), one olefinic proton at GH 6.16 (1H, br s, H-18), and four oxygenated methine
protons at GH 3.38 (1H, dd, J = 11.5, 4.5 Hz, H-3), 4.02 (1H, m, H-19), 4.63 (1H, m, H-12), and 5.08 (1H,
13
m, H-21) (Table 2). The C NMR spectrum of 3 exhibited 30 carbon signals, including seven methyl
carbons at δ 10.6, 17.0, 17.2, 19.3, 21.9, 22.6, and 28.6, two olefinic carbons at δ 120.4 and 150.9, four
oxygenated methines at δc 68.6, 69.6, 78.1, and 88.8, one carbonyl carbon at δc 179.7, three methines δc
56.4, 50.2, and 46.0, together with one sugar signals at δc 107.9, 73.3, 75.0, 69.9, and 67.2 (Table 1).
These data implied that the aglycone part of 3 could be a 3,18,19,21-terthydroxy-18,19-seco-ursan
derivative.7 Additionally, The 1H–1H COSY correlations of H-21 to H-22, H-20, and H-20 to H-30, H-19
and the HMBC cross-peaks of H-18 with C-17, C-22 and H-22 with C-28, and H-21 with C-22, C-20
provided the information of connection from γ-lactone ring moiety through the spiro carbon at C-17.
Consequently, the structure of E ring was elucidated as spiro-γ-lactone at C-17 by the signals of GC 179.7
(C-28), 78.1 (C-21), 46.1 (C-17), 43.7 (C-22) in the 13C NMR spectrum.
The sugar unit was identified as L-arabinopyranose by the signals of GH 4.72 (d, J = 7.0 Hz), 4.45 (m,
H-2'), 4.33 (m, H-4'), 4.32 (m, Hb-5'), 4.18 (m, H-3'), and 3.83 (m, Ha-5') in the 1H NMR spectrum and GC
107.9, 75.0, 73.3, 69.9, and 67.2 in the 13C NMR spectrum.15,16 The coupling constant (J = 7.0 Hz) of the

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HETEROCYCLES, Vol. 91, No. 6, 2015
anomeric proton signal at GH 4.72 indicated to be α-form.15,16 The sugar position was determined by
HMBC experiment, in which the long-range correlation was observed between the H-1' (δ 4.72) of
1
13
1
L-arabinopyranose and the C-3 (δ 88.8) of aglycone (Figure. 2). The H and C NMR, HMQC, H-1H
COSY, HMBC and ROESY spectra of 3 were almost same with those of dunnianaolactone B17, which
was isolated from Ilex dunniana, except the signals correlated to a sugar unit, arabinose. The relative
configurations of 3 were identified by analysis of ROESY data and comparison of the previous reported
data, indicating that the H-3, H-5, H-9, H-12, Me-23, and Me-27 were α-orientation and Me-24, Me-25,
and Me-26 were β-orientation. (Figure. 2). The ROESY cross-peaks of H-22α (δ 2.28) /H-21 (δ 5.08), and
H-19 (δ 4.02) /H-21 (δ 5.08) showed the same orientation for H19, H-21, and H-22α. The sugar,
α-L-arabinopyranose was confirmed by GC analysis and optical rotation after acid hydrolysis.14,18 Thus,
the structure of 3 was established as (3β,12β)-3-O-α-L-arabinopyranosyl-12,19-dihydroxy-18,19-ꢀ
secours-13(18)-ene-28,21-lactone.
Compound 4 was obtained as a colorless gum, and its molecular formula C41H66O15 was inferred from the
1
13
positive ion HRFAB-MS m/z 803.4190 [M+ Na]+ (calcd. for C41H64NaO14 803.4194). The H and C
NMR spectra of 4 were almost same with those of compound 3, except for the presence of a ketone group
at C-19. The position of ketone group was determined at C-19 on the basis of the HMBC correlations
1
between a H-30 (δ 1.02) of methyl group to C-19 (δ 209.0). The H–1H COSY correlations of H-21 to
H-22, H-20, and H-20 to H-30 and the HMBC cross-peaks of H-18 with C-17, C-22 and H-22 with C-28,
and H-21 with C-22, C-20 provided the information of connection from γ-lactone ring through the spiro
carbon at C-17. Consequently, the structure of E ring was elucidated as spiro-γ-lactone at C-17 by the
13
signals of GC 178.7 (C-28), 77.3 (C-21), 45.3 (C-17), 43.3 (C-22) in the C NMR spectrum. Also, the
1
13
analysis of H and C NMR spectra of 4 revealed the presence of arabinose and glucose units (Table 1
and 2). The configurations of the sugars were determined through the coupling constants of the anomeric
protons to be α-arabinopyranose (δH 4.99, d, J = 5.5 Hz), and β-glucopyranose (δH 5.19, d, J = 7.5 Hz).
The positions of two sugars were confirmed by the HMBC correlations between H-1' (δ 4.99) of
arabinopyranose to C-3 (δ 88.7) of aglycone, and between H-1'' (δ 5.19) of glucopyranose to C-2' (δ 81.0)
of arabinopyranose. The relative configurations of 4 were identified by the ROESY data and a
comparison with previously reported data, indicating that the H-3, H-5, H-9, H-12, Me-23 and Me-27
were α-oriented and Me-24, Me-25, and Me-26 were β-oriented (Figure. 2). The ROESY cross-peaks of
H-22α (δ 2.24) /H-21 (δ 4.82) showed the same orientation for H-21, and H-22α. The sugar,
α-L-arabinose and β-D-glucose in 4 were confirmed by GC analysis and optical rotation after acid
hydrolysis.14,18 Therefore, the structure of 4 was determined as (3β,12β)-3-[β-D-glucopyranosyl-
(1→2)-α-L-arabinopyranosyl]-12-hydroxy-19-oxo-18,19-secours-13(18)-en-28,21-lactone.

HETEROCYCLES, Vol. 91, No. 6, 2015
1193
HO
HO
HO
O
O
H
H
O
OH
H
H
O
O
OH
O
OH
HO
HO
O
O
OH
OH
HO
OH
2
O
OH
1
O
O
OH
O
OH
H
H
O
O
HO
O
H
H
H
H
H
H
H
O
O
HO
O
OH
HO
O
OH
OH
O
HO
4
3
O
OH
HO
OH
Figure 2. Key HMBC (
) and NOESY (
) correlations of 1-4
The known compounds were identified as cornutaoside A (5),7 ilexasprellanoside C (6),19 ilexoside G
(7),16 zigu-glucoside I (8),5 ilexside II (9),4 mateside (10),9 ilexoside XXVII (11),20 ilexoside XXX (12),21
brevicuspisaponin 3 (13),13 bifinoside A (14),22 ursolic acid (15),23 oleanolic acid (16),23 and
3β-Hydroxy-24-nor-urs-4(23),12-dien-28-oic acid (17)6 by comparison their NMR data with those
reported in the literature values.
The cytotoxic activities of the isolated compounds (1-17) a evaluated by determining their inhibitory
effects on human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and HCT15) in vitro using the
sulforhodamine B (SRB) assay.24 Compounds 16 and 17 showed moderate cytotoxic activity against the
four human tumor cell lines [IC50 (16): 12.57, 17.76, 12.92, 13.01 μM, IC50 (17): 12.92, 10.03, 7.82,
11.08 μM], respectively. But the other compounds were inactive (IC50: >30.0 μM).

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HETEROCYCLES, Vol. 91, No. 6, 2015
EXPERIMENTAL
General. Melting points are determined on a Gallenkamp melting point apparatus and are uncorrected. IR
spectra were recorded on a Bruker IFS-66/S FT-IR spectrometer. NMR spectra were recorded on a Varian
UNITY INOVA 500 NMR spectrometer operating at 500 MHz (1H) or 125 MHz (13C), with chemical
shifts given in ppm (δ). HR-ESI mass spectra were recorded on a SI-2/LCQ DecaXP Liquid
chromatography (LC)-mass spectrometer. HR-FAB mass spectra were obtained on a JEOL JMS700 mass
spectrometer. GC (Gas Chromatography) was carried out on using a HP-5 capillary column (30 cm ×
o
o
0.25 mm × 0.25 μm); column temperature, 230 C; injection temperature, 280 C; carrier gas, He.
Preparative HPLC was performed using a Gilson 306 pump with a Shodex refractive index detector.
Chromatographic separation was performed on an Econosil RP-18 10 μ column (250 × 10 mm i.d.).
Silica gel 60 (Merk Co., 70 - 230 mesh), RP-C18 silica gel (YMC GEL ODS-A, 12 nm, S-75 μm) and
Sephadex LH-20 (Pharmacia) were used for column chromatography. TLC was performed using Merck
pre-coated Silica gel F254 plates and RP-18 F254s plates. Low-pressure liquid chromatography was
performed over a LiChroprep Lobar-A RP-18 (240 × 10 mm i.d.) column with a FMI QSY-O pump
(ISCO).
Plant material. The leaves and trunks of Ilex cornuta (5.0 kg) were collected on Jeju Island, Korea in
March 2011, and the plant was identified by one of the authors (K.R.Lee). A voucher specimen
(SKKU-NPL 1108) has been deposited at the herbarium in the School of Pharmacy, Sungkyunkwan
University, Suwon, Korea.
Extraction and isolation. The leaves and trunks of I. cornuta (5.0 kg) were extracted with 80% aqueous
MeOH under reflux and filtered. The resulting MeOH extracts (550 g) were suspended in distilled water
(800 mL × 3) and then successively partitioned with n-hexane, CH2Cl2, EtOAc and n-BuOH, yielding 20,
68, 28 and 100 g, respectively. The EtOAc soluble fraction (28 g) was chromatographed on a silica gel
(230–400 mesh, 600 g) column eluted with CHCl3 - MeOH (40:1 – 1:1, gradient system) to yield eleven
fractions (E1–E11). Fraction E1 (7 g) was loaded on a Sephadex LH-20 column (450 g, 3 × 90 cm) and
eluted with CH2Cl2 – MeOH (1:1, 2 L) to yield three subfractions E11 - 13. Subfraction E12 (5 g) was
purified by semi-preparative reversed-phase HPLC using a 250 mm × 10 mm i.d., 10 μm, Econosil RP-18
column (Alltech) with a solvent system of 90% MeOH (2 L, flow rate; 2 mL/min) to give 15 (2 g), 16 (20
mg), and 17 (4 mg). Fraction E6 (500 mg) was loaded on a Sephadex LH–20 column (450 g, 3 × 90 cm)
and eluted with CH2Cl2 – MeOH (1 : 1) to yield four subfractions E61 – E64. Subfraction E63 (30 mg)
was separated by semi-preparative reversed-phase HPLC with a solvent system of 50% MeOH (1 L, flow

HETEROCYCLES, Vol. 91, No. 6, 2015
1195
rate; 2 mL/min) to give 6 (10 mg). Fraction E7 (5 g) was chromatographed on an RP–C18 silica gel
(230-400 mesh, 200 g) column chromatography (200 g, 3 × 30 cm) using a solvent system of 70% MeOH
(3 L) to give five subfractions (E71–E75). Subfraction E73 (250 mg) was purified by semi-preparative
reversed-phase HPLC with a solvent system of 50% MeCN (1 L, flow rate; 2 mL/min) to give 1 (70 mg),
2 (7 mg), and 10 (10 mg). Subfraction E74 (50 mg) was purified by preparative reverse-phase HPLC
(50% MeCN) to give 14 (5 mg). Fraction E8 (5 g) was loaded on a Sephadex LH-20 column (450 g, 3 ×
90 cm) and eluted with CH2Cl2- MeOH (1:1, 2 L) to yield four subfractions E81 – E84. Subfractions E82
(3 g) was chromatographed on an RP–C18 silica gel (230-400 mesh, 10 g) column chromatography (200 g,
3 × 30 cm) using a solvent system of 50% MeOH (2.5 L) to give five subfractions (E821–E825).
Subfractions E825 (500 mg) was purified by semi-preparative reversed-phase HPLC with a solvent
system of 20% MeCN (1 L, flow rate; 2 mL/min) to yield 8 (120 mg), 12 (50 mg), and 13 (15 mg).
Fraction E9 (850 mg) was chromatographed on an LiChroprep Lobar–B RP–18 column (using 40%
MeOH as eluant) and then purified by semi-preparative reversed-phase HPLC with a solvent system of
30% MeCN (1 L, flow rate; 2 mL/min) to yield 3 (7 mg), 4 (8 mg), and 5 (15 mg). Fraction E10 (1 g) was
loaded on a Sephadex LH–20 column (200 g, 3 × 30 cm) and eluted with CH2Cl2 – MeOH (1:1, 2 L) to
yield two subfractions E101 – 102. Subfractions E101 (70 mg) was purified by by semi-preparative
normal-phase HPLC using a solvent system of CHCl3/MeOH (7:1, 2 L, flow rate: 2 mL/min) to yield 9
(12 mg) and 11 (20 mg). Subfractions E102 (50 mg) was purified by semi-preparative reversed-phase
HPLC with a solvent system of 40% MeOH (1 L, flow rate; 2 mL/min) to afford 7 (7 mg).
Compound (1): a colorless gum; [α]2D5 +35.0 (c=2.0, MeOH); IR (KBr) νmax 3359, 2945, 2833, 1666,
1
13
1452, 1032, 649 cm-1; H-NMR data, see Table 2; C-NMR data, see Table 1; FABMS m/z 643 [M +
Na]+; HRFABMS m/z 643.3825 [M + Na]+; (calcd. for C35H56O9Na 643.3822).
Compound (2): a colorless gum; [α]2D5 +20.5 (c=0.3, MeOH); IR (KBr) νmax 3383, 2944, 2834, 1748,
1
13
1650, 1453, 1261, 1032, 652 cm-1; H-NMR data, see Table 2; C-NMR data, see Table 1; HRESI-MS
m/z 831.4500 [M + Na]+ (calcd. for C43H68NaO14, 831.4507).
Compound (3): a colorless gum; [α]2D5 -20.0 (c=0.2, MeOH); IR (KBr) νmax 3384, 2945, 2833, 1735,
1
13
1658, 1452, 1032, 655 cm-1; H-NMR data, see Table 2; C-NMR data, see Table 1; HRESI-MS m/z
643.3820 [M + Na]+ (calcd for C35H56O9Na, 643.3822).
Compound (4): a colorless gum; [α]2D5 -30.0 (c=0.4, MeOH); IR (KBr) νmax 3383, 2944, 2834, 1736,
1
13
1650, 1453, 1032, 652 cm-1; H-NMR data, see Table 2; C-NMR data, see Table 1; HRESI-MS m/z
803.4190 [M + Na]+ (calcd. for C41H64O14Na 803.4194).
Acid hydrolysis of compound 1-4 and sugar determination. Compound 1 (2mg) was dissolved in 3 mL

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HETEROCYCLES, Vol. 91, No. 6, 2015
of 1 N HCl, respectively. The solution was heat at 90 oC for 1 h. The hydrolysis, this reaction mixture was
diluted with H2O and extracted with CHCl3. The H2O layer was dried in vacuo, and stirred with pyridine
o
(0.1 mL) and L-cysteine methyl ester hydrochloride (2.0 mg). The mixture was stirred at 60 C for 1.5 h.
After the reaction mixture was dried in vacuo, the residue was trimethylsilylated with
1-trimethylsilylimidazole (0.1 mL) for 2 h. The mixture was partitioned between n-hexane and H2O (0.3
mL, each), and the organic layer (1 μL) was analyzed by GC.25,26 Compounds 2-4 (each, 2 mg) were
treated using the same method. D-glucose and L-arabinose were treated in the same manner. L-arabinose
(1 : 5.42 min, 3 : 5.45 min) was detected in 1, and 3. D-glucose (2 : 9.74 min, 4 : 9.77 min) and
L-arabinose (2 : 5.41 min, 4 : 5.44) were detected in 2, and 4. Identification of D-glucose (9.76 min) and
L-arabinose (5.48 min) were detected in each case by co-injection of the hydrolysate with standard
silylated sugars.
Cytotoxicity Assay. A sulforhodamine B bioassay (SRB) was used to determine the cytotoxicity of the
above 17 compounds. The cytotoxic activity of each compound against four cultured human tumor cells
was examined in vitro at the Korea Research Institute of Chemical Technology. The tumor cell lines were
A549 (non small cell lung adenocarcinoma), SK-OV-3 (ovarian cancer cells), SK-MEL-2 (skin
melanoma) and HCT15 (colon cancer cells).24 Doxorubicin was used as the positive control. IC50 values
for the cytotoxicity of etoposide were 0.25, 1.73, 0.14, and 3.36 μM to A549, SK-OV-3, SK-MEL-2, and
HCT15 cells, respectively.
ACKNOWLEDGEMENTS
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 Technology
(2012R1A5A2A28671860). We are thankful to the Korea Basic Science Institute (KBSI) for the
measurements of MS spectra.
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