106
N.-L. Song et al. / Phytochemistry Letters 13 (2015) 103–107
KIB20050623) was deposited at the Laboratory of Phytochemistry,
Kunming Institute of Botany, Chinese Academy of Sciences.
3.3. Extraction and isolation
The dried and powdered tubers of H. chinensis (3.84 kg) were
extracted with MeOH (7 L ꢂ 6, each 8 h) at 60 ꢁC. After removal of
the solvent under reduced pressure, a residue (1.21 kg) was
obtained. This residue was dissolved in H2O (3 L) and then
partitioned successively with EtOAc (2 L ꢂ 3) and n-BuOH (2 L ꢂ 3)
to give EtOAc extract (220.10 g) and n-BuOH extract (109.40 g). The
n-BuOH ext. was subjected to silica gel column eluted with a
gradient of CHCl3/MeOH (20:1, 10:1, 5:1, 80 L each) and CHCl3/
MeOH (0:1, 8.0 L) to afford four Fractions a-d. Fr. d (10.4 g) was
chromatographed on RP-18 column eluted with MeOH/H2O
(40:60) to yield Fr.d-1 (1.4 g) and 3 (11.3 mg). The Fr.d-1 was
separated on silica gel column eluted with CHCl3/MeOH (4:1) to
obtain 1 (13.6 mg) and 2 (21.0 mg).
3.3.1. Hemsloside-Ma4 (1)
White powder; ½a D
ꢃ
18 + 18.52 (c = 0.2, MeOH); IR (KBr) nmax
:
3273, 3173, 2915,1735,1654, and 1460 cmꢀ1; 1H NMR and 13C NMR
data see Tables 1 and 2; HR-FAB-MS [M + Na]+ ion peak at m/z
1125.5437 (C54H86O23; calcd. for 1125.5458); FAB-MS (negative):
m/z 1102 [M]ꢀ (60), 940 (10), 777 (65), 455 (25).
3.3.2. Hemsloside-Ma5 (2)
White powder; ½a D21
ꢃ
ꢀ 6.59 (c = 1.8, MeOH); IR (KBr) nmax: 3273,
Fig. 4. The key HMBC (H ! C) and NOESY ($) correlations of compound 3.
3173, 2915, 1735, 1654, and 1460 cmꢀ1; 1H NMR and 13C NMR data
see Tables 1 and 2; HR-FAB-MS [M]ꢀ ion peak at m/z 1072.5443
(C53H84O22; calcd. for 1072.5454); FAB-MS (negative): m/z 1072
[M]ꢀ (30), 940 (5), 777 (65), 645 (25), 455 (25).
hepatocellular carcinoma), MCF7 (human breast adenocarcinoma),
HT29 (human colonic adenocarcinoma) and MKN28 (human
gastric cancer metastasis) cell lines and cisplatin was positive
control in our assay (see Supplementary data).
3.3.3. Hemsloside-Ma6 (3)
White powder; ½a D21
ꢃ
ꢀ 58.96 (c = 1.4, MeOH); IR (KBr) nmax
:
3. Experimental
3434, 3287, 2846, 1727, and 1451 cmꢀ1; 1H NMR and 13C NMR data,
see Table 3; HR-FAB-MS [M + Na]+ ion peak at m/z 797.3914
(C38H62O16Na; calcd. 797.3936); FAB-MS (negative): m/z 773 [M-
1]ꢀ (90), 627 [M-Rha]ꢀ (5), 465 [M-Rha-Glc]ꢀ (100), 303 [M-Rha-
Glc-Rha]ꢀ (3).
3.1. General experimental procedure
Methanol (HPLC grade) was purchased from Fisher Chemicals
(New Jersey, USA). NMR spectra were recorded with a Bruker DRX-
500 spectrometer using Me4Si as an internal standard. MS was
measured at an Agilent 1100 LC/MSD TOF spectrometer. The water
3.4. Acid hydrolysis of compounds 1–3
was purified with
a purity water system (Chengdu YouPu
Electronic Products Technology Co., Ltd., China and the system
model name is UPTL-1-100L). Silica gel (200–300 mesh), RP-18 and
Sephadex LH-20 for column chromatography were supplied by the
Merck Corporation (Germany). GF254 plates (Qingdao Marine
Chemical Corporation, China) were used for TLC, and Spots were
visualized under UV light or by spraying with 10% H2SO4 in ethanol
The experiment was implemented using the method previously
described (Li et al., 2009). Compounds 1–3 (2.0 mg) were treated
with 3% HCl in MeOH (5 mL) at 92 ꢁC for 3 h, respectively. HCl and
MeOH were removed under reduced pressure at 60 ꢁC. 5 mL CHCl3/
H2O (1:1) were used for extraction. The aqueous layers were
evaporated to dryness and analyzed by TLC over silica gel (CHCl3–
MeOH–H2O–HCOOH 15:5:1:1) by comparison with authentic
followed by heating.
D-Xylose, D-glucose, L-rhamnose and L-
arabinose were purchased from Sigma (USA) and New Jersey
(USA), respectively. The human HepG2, MCF7, HT29 and MKN28
cancer cell lines were obtained from the American Type Culture
Collection (Manassas, VA, USA), and cultured in RPMI 1640 media
(Gibco, SanFrancisco, CA, USA) containing 10% fetal bovine serum
(FBS), penicillin (100 U/mL), and streptomycin (100 g/mL). The cells
were maintained at 37 ꢁC in an atmosphere of 5% CO2 and 95% air
according to standard. Microplate reader (Bio-Tek Winooski,
Vermont, USA) was employed to read OD date.
samples (
L
-arabinose Rf 0.75;
L-rhamnose Rf 0.65; D-xylose Rf
0.69; -glucose Rf 0.55;
D
D
-glucuronic acid Rf 0.10). The results
further confirmed that the structure of compounds 1–3.
3.5. Cytotoxic activity assay
The assay was carried out using the method previously
described (Nian et al., 2010). About 1 ꢂ104 cell/well were seed
into 96-well microtiter plates, after twenty-four hours post-
seeding cells were treated with vehicle control or various
3.2. Plant material
concentrations of samples for 48 h, 20 mL of MTT solution (5 mg/
mL, Sigma–Aldrich, St. Louis, MO, U.S.A) was added to each well
and the tumor cells were incubated at 37 ꢁC in a humidified
atmosphere of 5% CO2 air for 4 h. Upon removal of MTT/medium,
The tubers of H. chinensis were collected at Dongchuan County,
Kunming city, Yunnan Province of China, in October 2004. It was
identified by Prof. Shu-kun Chen, and a dry specimen (No.
150 mL of DMSO was added to each well and the plate was agitated