Journal of Natural Products
Note
icariside E4 (63 mg). Further purification of fraction A3a by
semipreparative HPLC (MeOH−H2O, 40:60, 3 mL/min) yielded
compound 2 (6 mg).
with 3. The β-glycosidic linkage and D-configurations of apiose
and glucose were determined by the same methods as those
of 3. The structure of 4 was thus elucidated as 5-[O-β-D-
apiofuranosyl-(1→2)-O-β-D-glucopyranosyl] methyl gentisate.
Eight known phenolic glycosides were also isolated and
identified as 3,4,5-trimethoxyphenyl-1-O-β-D-apiofuranosyl-
(1→6)-O-β-D-glucopyranoside,12 4-hydroxymethyl-2-methoxy-
phenyl-1-O-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyrano-
side,13 icariside E4,14 syringaresinol-β-D-glucopyranoside,15 3,5-
dimethoxy-4-hydroxybenzyl alcohol-4-O-β-D-glucopyranoside,16
koaburside,17 vanilloloside,18 and scopolin19 on the basis of their
spectroscopic data (1H NMR, 13C NMR, and ESIMS).
All compounds isolated were tested for their cytotoxicity
effects against two human colon tumorigenic (HCT-116 and
Caco-2) cell lines and a nontumorigenic (CCD-18Co) cell line.
However, none of the compounds proved to be cytotoxic for
any of these cell lines (IC50 < 5 μg/mL).
The n-butanol extract (117.0 g) was subjected to an XAD-16
Amberlite resin column (8 × 35 cm) eluting with MeOH−H2O
(20:80 to 100:0) to give four fractions (1−4). Fraction 2 was subjected
to CC eluted with chloroform−methanol (10:1 to 1:1) in gradient
to obtain three fractions (2A−2C). Purification of fraction 2A by
semipreparative HPLC (MeOH−H2O, 30:70 to 50:50, in 25 min,
3 mL/min) gave syringaresinol-β-D-glucopyranoside (15 mg). Fraction
2C was purified on a column of Sephadex LH-20 eluted with MeOH
to give scopolin (20 mg) and subfraction 2C1. Subfraction 2C1 was
separated over MPLC, by elution with MeOH−H2O (10:90 to 70:30,
3 mL/min), to obtain five fractions (2C1a−2C1e). Fraction 2C1b was
subjected to semipreparative HPLC (MeOH−H2O, 10:90 to 35:65 in
14 min, 3 mL/min), to yield 3,5-dimethoxy-4-hydroxybenzyl alcohol
4-O-β-D-glucopyranoside (8 mg) and 4-hydroxymethyl-2-methoxy-
phenyl 1-O-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside (3 mg).
Purification of 2C1c by semipreparative HPLC (MeOH−H2O, 23:77,
3 mL/min) gave 3,4,5-trimethoxyphenyl 1-O-β-D-apiofuranosyl-(1→
6)-O-β-D-glucopyranoside (10 mg). Fraction 2C1d was separated
using semipreparative HPLC (MeOH−H2O, 10:90 to 50:50 in
18 min, 3 mL/min) and yielded 3 (8 mg) and 4 (4 mg). Fraction
2C1e was purified by semipreparative HPLC (MeOH−H2O, 10:90 to
40:60 in 12 min, 3 mL/min) to yield vanilloloside (8 mg).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on an Auto Pol III automatic polarimeter (Rudolph
Research, Flanders, NJ, USA) at room temperature. The IR spectra
were recorded on a Nicolet 380 FT-IR spectrometer. The UV spectra
were measured on a Shimadzu UV-2550 UV−visible spectropho-
tometer. 1D and 2D NMR data were recorded on a Varian 500 MHz
instrument with TMS as internal standard. HRESIMS data were
acquired using an LTQ Orbitrap XL mass spectrometer (Thermo
Scientific). Semipreparative HPLC separations were performed on a
Hitachi Elite LaChrom system consisting of an L2130 pump, an
L-2200 autosampler, an L-2455 diode array detector, and a
Phenomenex Luna C18 column (250 × 10 mm, S-5 μm), all operated
by EZChrom Elite software. Medium-pressure liquid chromatography
(MPLC) separations were carried out on prepacked C18 columns (4 ×
37 cm) connected to a DLC-10/11 isocratic liquid chromatography
pump (D-Star Instruments, Manassas, VA, USA) with a fixed-
wavelength detector. All solvents were of ACS or HPLC grade and
were obtained from Sigma-Aldrich (St. Louis, MO, USA) through
Wilkem Scientific (Pawcatuck, RI, USA). Silica gel (230−400 mesh,
Sorbent Technologies), Sephadex LH-20 gel (Amersham Biosciences),
and MCI gel (CHP20P, 63−150 μM, M & M Industries Inc.) were
used for column chromatography, and precoated silica gel GF254
plates (Whatman Ltd., Maidstone, England) were used for TLC
analysis. Samples of D-glucose, D-apiose, MTS salt [3-(4,5-dimethyl-
thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfenyl)-2H-tetrazo-
lium salt], and etoposide were purchased from Sigma-Aldrich (St.
Louis, MO, USA).
Saccharumoside A (1): colorless, amorphous solid; [α]20 −31
D
(c 0.5, MeOH); UV (MeOH) λmax (log ε) 293 (3.45), 267 (3.73), 223
1
(3.91) nm; IR νmax 3344, 2968, 1691, 1608, 1496, 1043 cm−1; H
NMR and 13C NMR data, see Table 1; HRESIMS m/z 671.2314
[M − H]− (calcd for C34H39O14, 671.2340).
Saccharumoside B (2): colorless, amorphous powder; [α]20 +75
D
(c 0.2, MeOH); UV (MeOH) λmax (log ε) 299 (3.56), 265 (3.78), 220
1
(3.99) nm; IR νmax 3415, 2935, 1699, 1598, 1500, 1064 cm−1; H
NMR and 13C NMR data, see Table 1; HRESIMS m/z 465.1395
[M − H]− (calcd for C22H25O11, 465.1397).
Saccharumoside C (3): colorless, amorphous solid; [α]20 −86
D
(c 0.5, MeOH); UV (MeOH) λmax (log ε) 320 (3.69), 234 (3.92) nm;
1
IR νmax 3375, 1701, 1601, 1501 cm−1; H NMR and 13C NMR data,
see Table 2; HRESIMS m/z 507.1351 [M + HCOO]− (calcd for
C20H27O15, 507.1350).
Saccharumoside D (4): colorless, amorphous solid; [α]20 −13
D
(c 0.4, MeOH); UV (MeOH) λmax (log ε) 321 (3.69), 236 (3.87) nm;
1
IR νmax 3430, 1698, 1603, 1495 cm−1; H NMR and 13C NMR data,
see Table 2; HRESIMS m/z 507.1352 [M + HCOO]− (calcd for
C20H27O15, 507.1350).
Acid Hydrolysis of Compounds 1−4 and Sugar Analysis.
Each compound (2 mg) was added to a mixture of concentrated HCl
(0.5 mL), H2O (1.5 mL), and dioxane (3 mL) and refluxed for 2 h.
After completion of the reaction (monitored by TLC), H2O was
added to the reaction mixture, which was then extracted with CHCl3
(3 × 5 mL). The aqueous layer was neutralized with NaHCO3 and
then concentrated to dryness under reduced pressure and purified by
Sephadex LH-20 chromatography to give a sugar fraction. The sugar
fraction was analyzed by HPLC under the following conditions:
column, Waters Xbridge Amide (100 × 4.6 mm, 3.5 μm); column
temperature, 35 °C; mobile phase, acetonitrile−water (75/25, v/v)
with 0.2% TEA; flow rate, 1.5 mL/min; detector, refractive index
(35 °C). Identification of D-glucose and D-apiose was carried out by
comparison of these retention times and optical rotations with those
of authentic samples. D-Glucose: tR 2.4 min, positive optical rotation;
D-apiose: tR 1.5 min, positive optical rotation.
Cytotoxicity Assay. Two human colon cancer cell lines, Caco-2
(adenocarcinoma) and HCT-116 (carcinoma), and the nontumori-
genic colon cell line CCD-18Co were obtained from American Type
Culture Collection (Rockville, MD, USA). Caco-2 and CCD-18Co
cells were grown in EMEM medium supplemented with 10% v/v fetal
bovine serum, 1% v/v nonessential amino acids, 1% v/v L-glutamine,
and 1% v/v antibiotic solution (Sigma). HCT-116 cells were grown in
McCoy’s 5A medium supplemented with 10% v/v fetal bovine serum,
1% v/v nonessential amino acids, 2% v/v HEPES, and 1% v/v
Plant Material. The bark of A. saccharum was collected in the
summer of 2009 by the Federation of Maple Syrup Producers of Quebec
(Canada), shipped to our laboratory in August 2009, and identified by
Mr. J. Peter Morgan (Senior Gardener, College of Pharmacy, University
of Rhode Island). A voucher specimen (JPMCB1) has been deposited in
the Heber-Youngken Herbarium and Greenhouse, College of Pharmacy,
University of Rhode Island.
Extraction and Isolation. The air-dried powder of the bark
(4.2 kg) of A. saccharum was extracted by maceration with methanol
(10 L × 3 times for 7 days per time period) at room temperature to
afford 258.1 g of crude extract. The extract was suspended in distilled
water (1 L) and then extracted successively with ethyl acetate (1 L ×
3 times) and n-butanol (1 L × 3 times). The ethyl acetate fraction
(33.4 g) was chromatographed over a column (5 × 50 cm) of MCI gel
(MeOH−H2O, 50:50 to 90:10) to yield four fractions (A−D).
Fraction A (23.5 g) was subjected to silica gel chromatography (CC)
eluted with chloroform−methanol (20:1 to 2:1) in a gradient to obtain
three fractions (A1−A3). Fraction A3 was chromatographed over
a column (3 × 70 cm) of Sephadex LH-20 eluted with MeOH to
give four subfractions (A3a−A3d). Fraction A3a was separated by
semipreparative HPLC eluted with MeOH−H2O (20:80 to 80:20 in
30 min, 3 mL/min) to yield koaburside (10 mg), 1 (11 mg), and
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dx.doi.org/10.1021/np200678n|J. Nat. Prod. 2011, 74, 2472−2476