L. Li et al. / Phytochemistry Letters 6 (2013) 570–574
573
Table 5
3.2. Plant material
Inhibitory effects of compounds 1–7 against two colorectal human cancer cell lines
HCT-116 and HT-29.
The roots and rhizomes of C. mandshurica were collected in
September 2010 in Jinzhou city, Liaoning Province of China. The
identification of the plant was performed by one of the authors (Y.-
X. He). A voucher specimen (CM 201009) is maintained in the
herbarium of Bioengineering College of Xihua University.
Compound
IC50 value (
HCT-116
mM)
HT-29
1
3.0
0.6
2.8
0.9
2
3
2.7
4.2
4
12.3
8.4
10.3
7.3
3.3. Extraction and isolation
5
6
12.9
16.1
0.0035
13.5
12.5
0.0034
The dried roots and rhizomes (3 kg) of C. mandshurica were
extracted with 50% EtOH. After removing the solvent, the residue
(822 g) was suspended in H2O and extracted with n-BuOH. The n-
BuOH extract (93 g) was subjected to silica gel column chroma-
tography and eluted with CHCl3-MeOH (10:1–1:1, v/v) to afford
fractions 1–5. Fraction 1 (12 g) was subjected to C18 silica gel
column chromatography and eluted with MeOH–H2O in a gradient
of MeOH (MeOH–H2O, 10:90–100:0, v/v, to afford subfractions 1–1
to 1–4 (1.3 g, 1.7 g, 3.3 g, and 3.4 g, respectively). Subfraction 1–2
(1.7 g) was isolated by preparative HPLC (MeCN–H2O, 30:70, v/v,
2.0 mL/min) to yield compounds 1 (33 mg) and 4 (39 mg).
Subfraction 1–4 (3.3 g) was isolated by preparative HPLC
(MeCN–H2O, 31:69, v/v, 2.0 mL/min) to yield compounds 2
(33 mg) and 3 (39 mg). Fraction 5 (5.2 g) was subjected to C18
silica gel column chromatography and eluted with MeOH–H2O in a
gradient of MeOH (MeOH–H2O, 10:90–100:0, v/v, to afford
subfractions 5–1 to 5–3 (1.4 g, 2.1 g, and 1.1 g, respectively).
Subfraction 5–1 (1.4 g) was isolated by preparative HPLC (MeCN–
H2O, 32:68, v/v, 2.0 mL/min) to yield compounds 5 (28 mg) and 7
(47 mg). Subfraction 5–2 (2.1 g) was isolated by preparative HPLC
(MeCN–H2O, 31:69, v/v, 2.0 mL/min) to yield compound 6 (82 mg).
7
Paclitaxel
with Amberlite IRA-400 resin (OHꢀ form) and the resin was
filtered. After removal of the solvent under pressure from the
filtrate, the residue was passed through a Sep-Pak C18 cartridge
with H2O and MeOH. The H2O eluate was concentrated and the
residue was treated with L-cysteine methyl ester hydrochloride
(2.0 mg) in pyridine (1.0 ml) at 60 8C for 2 h. After dried by N2 gas,
the residue was treated with N-(trimethylsilyl)imidazol (0.2 ml) at
60 8C for 1 h. The reaction was ended by adding water (1.0 ml), and
extracted with cyclohexane (1.0 ml, three times). The cyclohexane
layer was collected and concentrated to 1.0 ml for GC analysis.
Separations were carried out on a HP-5 column (28 m ꢁ 0.32 mm).
High purity He was employed as carrier gas (1.0 ml/min flow rate),
and the FID detector operated at 260 8C (column temp 180 8C). The
retention times of the monosaccharide derivatives were as follows:
L
-Rha, 5.78 min;
The retention times of standard monosaccharide derivatives were:
-Rha, 5.77 min; -Glc, 11.87 min; -Rib, 5.41 min; -Ara, 5.10 min.
D-Glc, 11.85 min; D-Rib, 5.40 min; L-Ara, 5.11 min.
L
D
D
L
3.3.1. Mandshunosides C (1)
3.5. Alkaline hydrolysis
White amorphous solid, ½a d20
ꢀ52, (c = 1.0, MeOH); UV (MeOH)
ꢂ
l
max 203 nm; IR(KBr)
n
max (cm ꢀ1): 3412, 2931, and 1730; 1H NMR
Compound 2 (10 mg) was refluxed in 5% KOH solution (pH 12–
13) at 90 8C for 1 h. The reaction mixture was neutralized with 5%
HCl solution and then concentrated to dryness. The residue was
extracted with n-BuOH, and the organic layer analyzed by NMR
spectroscopy. Hydrolysis of 2 afforded 1.
(C5D5N, 500 MHz):
d 1.12 (3H, s, Me-27), 1.06 (3H, s, Me-24), 1.05
(3H, s, Me-26), 0.92 (3H, s, Me-25), 0.84 (3H, s, Me-29), 0.82 (3H, s,
Me-30), 4.26 (1H, dd, J = 12.0, 5.0 Hz, H-3), 4.22 (1H, d, J = 12.0 Hz,
H-23a), 3.86 (1H, d, J = 12.0 Hz, H-23b), 5.36 (1H, br s, H-12). 13C
NMR spectral data, see Tables 1 and 3.
3.6. MTT cytotoxicity assay
3.3.2. Mandshunoside D (2)
White amorphous solid, ½a d20
ꢂ
ꢀ31 (c = 1.0, MeOH); UV (MeOH)
max (cm
1.14
The bioassay was carried out according to a previously
l
max (log
e
) 242 (2.34), 294 (sh), 326 (4.24) nm; IR(KBr)
n
described method, against two human colorectal cancer cell lines
(HCT-116 and HT-29) (Carmichael et al., 1987). Paclitaxel was used
as positive control (Table 5).
ꢀ1): 3406, 2933, 1732, and 1630; 1H NMR (C5D5N, 500 MHz):
d
(3H, s, Me-27), 1.07 (3H, s, Me-24), 1.06 (3H, s, Me-26), 0.92 (3H, s,
Me-25), 0.85 (3H, s, Me-29), 0.83 (3H, s, Me-30), 4.27 (1H, dd,
J = 12.0, 5.0 Hz, H-3), 4.24 (1H, d, J = 12.0 Hz, H-23a), 3.86 (1H, d,
J = 12.0 Hz, H-23b), 5.35 (1H, br s, H-12); 13C NMR spectral data,
see Tables 1 and 3; 1H NMR and 13C NMR spectral data for
isoferuloyl moiety, see Table 4.
Acknowledgements
This work was supported by the National Natural Science fund
of China (Grant No. 81202897); the Chun Hui project of Ministry of
Education (Z2010102); the Chun Hui project of Ministry of
Education (Z2010103); the Open Research fund of Key Laboratory
of Food Biotechnology, Xihua University (SZJJ2012-006); and the
Innovation Fund of Postgraduate, Xihua University (YCJJ201243).
3.3.3. Mandshunoside E (3)
White amorphous solid, ½a d20
ꢀ37 (c = 1.0, MeOH); UV (MeOH)
ꢂ
l
max 203 nm; IR(KBr) n
max (cm ꢀ1): 3406, 2933, and 1731; 1H NMR
(C5D5N, 500 MHz):
d 1.15 (3H, s, Me-27), 1.08 (3H, s, Me-24), 1.07
(3H, s, Me-26), 0.94 (3H, s, Me-25), 0.86 (3H, s, Me-29), 0.85 (3H, s,
Me-30), 4.27 (1H, dd, J = 12.0, 5.0 Hz, H-3), 4.25 (1H, d, J = 12.0 Hz,
H-23a), 3.86 (1H, d, J = 12.0 Hz, H-23b), 5.37 (1H, br s, H-12); 13C
NMR spectral data, see Tables 1 and 3.
References
Carmichael, J., De Graff, W.G., Gazdar, A.F., Minna, J.D., Mitchell, J.B., 1987. Evalua-
Fu, Q., Zan, K., Zhao, M.B., Zhou, S.X., Shi, S.P., Jiang, Y., Tu, P.F., 2010. Triterpene
3.4. Acid hydrolysis
Jhoo, J.W., Sang, S.M., He, K., Cheng, X.F., Zhu, N.Q., Stark, R.E., Zheng, Q.Y., Rosen,
A
solution of compound 1 or 3 (5.0 mg each) in 2 M
trifluoroacetic acid (1 ml) was heated at 110 8C for 2 h, and then
dried by N2 gas. After cooling, the reaction mixture was neutralized