462 Letters
Compound
IC50 (µM)a
A549
Table 1 Cytotoxicity of com-
pounds 1–7 against four cultured
human cancer cell lines using the
SRB assay in vitro.
SK‑OV‑3
SK‑MEL‑2
HCT-15
1
> 100.0
> 100.0
> 100.0
24.29
22.64
62.64
12.41
0.16
> 100.0
> 100.0
> 100.0
26.72
27.19
57.19
17.62
0.38
> 100.0
> 100.0
> 100.0
11.08
24.11
70.11
11.73
0.04
> 100.0
> 100.0
> 100.0
29.20
16.42
46.42
14.29
0.82
2
3
4
5
6
7
Doxorubicinb
a The IC50 value was defined as the concentration (µM) that caused 50% inhibition of cell growth in vitro; b Doxorubicin was used as a
positive control
Compounds 2 (C16H26O7) and 3 (C16H26O7) showed an [M + H]+
peak at m/z = 331.1757 and 331.1774 (calcd. for C16H27O7:
331.1757), respectively, in the HR‑FAB‑MS. Their NMR spectral
data displayed the presence of one glucose, three tert-methyls,
two methylenes, one methine, two quaternary carbons, one hy-
droxylated methine, and one carbonyl carbon, which were simi-
lar to those of 5-hydroxycamphor [13]. From the results of the
HMBC experiment (HMBC between H-1′/C-5), 2 and 3 had a glu-
over silica gel (230–400 mesh, 500 g, 6 × 90 cm), eluting with a
gradient solvent system of CHCl3-MeOH‑H2O (4:1:0.1, 5:2:0.1,
and 5:2:0.2, 2 L of each solvent) to yield five crude fractions (F1–
F5). F2 (2.4 g) was applied to CC over Sephadex LH-20 (200 g,
3 × 90 cm; Pharmacia Co.), eluting with a solvent system of
MeOH‑H2O (9:1, 1.5 L) to give 8 subfractions (F21–F28). The sub-
fraction F23 (500 mg) was purified further by semipreparative
HPLC, using CH2Cl2-MeOH (13:1) over 30 min at a flow rate of
2.0 mL/min (Alltech Econosil Silica 5 µ column; 250 × 10 mm;
cosyl moiety at C-5. Because NOE interactions between H-3endo
/
H-5 and between H-5/H-6endo were observed in NOESY spectra
of 2, the configuration of H-5 of 2 should be endo. The CD spec-
trum of 2 showed a negative Cotton effect at 295 nm [7]. Thus, 2
was identified as (1S,4S,5S)-5-exo-hydroxycamphor 5-O-β-D-glu-
copyranoside. In addition, NOESY correlation of 3 between H-5/
H3-8 indicated that the configuration of H-5 of 3 should be exo.
Thus, 3 was characterized as (1R,4R,5S)-5-endo-hydroxycamphor
5-O-β-D-glucopyranoside, which was supported by a positive
Cotton effect at 294 nm in the CD spectrum [7].
Shodex refractive index detector) to yield 3 (12 mg, Rt =
15.5 min). The subfraction F25 (190 mg) was also purified by
semipreparative HPLC, using CHCl3-MeOH (10:1) to yield 2
(21 mg, Rt = 13.0 min). F3 (3.1 g) was subjected to CC over silica
gel (230–400 mesh, 500 g, 9 × 60 cm) using CHCl3-MeOH (4:1,
3 L) to yield 6 fractions (F31–F36). Compound 1 (10 mg) was iso-
lated from F32 (100 mg) by semipreparative HPLC using CHCl3-
MeOH (6:1, flow rate of 2.0 mL/min, Rt = 14.5 min). Compounds
4-7 were isolated from the n-hexane-soluble fraction (18 g) by
CC over silica gel, Sephadex LH-20, and semipreparative HPLC.
Amoxanthoside A (1): 10 mg; amorphous gum; [α]2D5: − 4.7 (c 0.2,
MeOH); IR (KBr): νmax = 3390, 2947, 1638, 1026 cm−1; FAB‑MS:
m/z = 789 [M + H]+; HR‑FAB‑MS: m/z = 789.3925 [M + H]+ (calcd.
In this study, the cytotoxicity of the isolates (1-7) against A549,
SK‑OV‑3, SK‑MEL‑2, and HCT15 human tumor cell lines was eval-
uated using the sulforhodamine B (SRB) assay in vitro. The results
"
(l Table 1) showed that 4, 5, and 7 exhibited cytotoxicity against
for C38H61O17: 789.3909). 1H- and 13C‑NMR data: see l Table 2.
"
A549, SK‑OV‑3, SK‑MEL‑2, and HCT15 cells, while the other com-
pounds showed little cytotoxicity against the tested cell lines
(IC50 > 30 µM).
(1S,4S,5S)-5-exo-hydroxycamphor 5-O-β-D-glucopyranoside (2):
21 mg; colorless gum; [α]D25: − 20.8 (c 0.8, MeOH); IR (KBr):
ν
max = 3394, 2965, 1728, 1639, 1371, 1028 cm−1; CD (MeOH):
Materials and Methods
!
λmax (Δε) = 295 (− 2.6) nm; FAB‑MS: m/z = 331 [M +
H]+;
HR‑FAB‑MS: m/z = 331.1757 [M + H]+ (calcd. for C16H27O7:
331.1757). 1H‑NMR (500 MHz, CD3OD): δ = 4.30 (1H, d, J = 8.0 Hz,
H-1′), 4.03 (1H, dd, J = 3.5, 7.5 Hz, H-5endo), 3.91 (1H, br d,
J = 11.5 Hz, H-6′a), 3.72 (1H, dd, J = 3.5, 11.5 Hz, H-6′b), 3.37–3.14
(4H, m, H-2′, 3′, 4′, 5′), 2.47 (1H, dd, J = 1.0, 5.0 Hz, H-4), 2.36 (1H,
dd, J = 5.0, 18.5 Hz, H-3exo), 1.95 (1H, dd, J = 7.5, 15.0 Hz, H-6endo),
1.78 (1H, m, H-6exo), 1.76 (1H, d, J = 18.5 Hz, H-3endo), 1.23 (3H, s,
H-8), 0.90 (3H, s, H-10), 0.84 (3H, s, H-9); 13C‑NMR (125 MHz,
CD3OD): δ = 221.1 (C-2), 103.9 (C-1′), 82.7 (C-5), 78.4 (C-5′), 78.1
(C-3′), 75.2 (C-2′), 71.8 (C-4′), 62.9 (C-6′), 59.5 (C-1), 50.5 (C-4),
47.7 (C-7), 40.9 (C-6), 39.6 (C-3), 21.2 (C-9), 20.7 (C-8), 9.4 (C-10).
(1R,4R,5S)-5-endo-hydroxycamphor 5-O-β-D-glucopyranoside (3):
12 mg; colorless gum; [α]D25: − 14.8 (c 0.4, MeOH); IR (KBr):
The seeds of A. xanthioides (2.5 kg), which were imported from
China, were bought at Kyungdong Market in December 2007
and identified by one of the authors (K.R.L.). A voucher specimen
(SKKU-2007–12B) of the plant was deposited at the College of
Pharmacy at Sungkyunkwan University, Suwon, Korea. NMR
spectra, including 1H-1H COSY, HMQC, HMBC, and NOESY experi-
ments, were recorded on a Varian UNITY INOVA 500 NMR spec-
trometer operating at 500 MHz (1H) and 125 MHz (13C). Optical
rotations were measured on a Jasco P-1020 polarimeter in MeOH.
IR spectra were recorded on a Bruker IFS-66/S FT‑IR spectrome-
ter. CD spectra were measured on a JASCO J-715 spectropolarim-
eter. FAB and HR‑FAB mass spectra were obtained on a JEOL
JMS700 mass spectrometer.
The seeds of A. xanthioides (2.5 kg) were extracted at room tem-
perature with 80% MeOH and evaporated under reduced pres-
sure to give a residue (210 g), which was dissolved in water
(800 mL) and partitioned with solvent to give n-hexane- (18 g),
CHCl3- (11 g), and n-BuOH- (23 g) soluble portions. The n-BuOH-
soluble fraction was subjected to column chromatography (CC)
ν
max = 3393, 2966, 1730, 1638, 1375, 1077 cm−1; CD (MeOH):
λmax (Δε) = 294 (+ 2.1) nm; FAB‑MS: m/z = 331 [M
H]+;
+
HR‑FAB‑MS: m/z = 331.1774 [M + H]+ (calcd. for C16H27O7:
331.1757). 1H‑NMR (500 MHz, CD3OD): δ = 4.73 (1H, dddd,
J = 2.0, 4.0, 4.5, 9.5 Hz, H-5exo), 4.28 (1H, d, J = 7.5 Hz, H-1′), 3.87
(1H, br d, J = 11.5 Hz, H-6′a), 3.67 (1H, dd, J = 3.5, 11.5 Hz, H-6′b),
3.35–3.14 (4H, m, H-2′, 3′, 4′, 5′), 2.80 (1H, d, J = 19.0 Hz, H-3endo),
Kim KH et al. Terpene Glycosides and… Planta Med 2010; 76: 461–464