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RSC Advances
Page 3 of 10
DOI: 10.1039/C6RA14467E
Journal Name ARTICLE
Extraction and isolation
Cell viability was determined using the 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as previously
described Zong et al.11 Briefly, cell lines in culture medium were
placed in a cell of a 96-well plate at a concentration of
4×103cells/mL and incubated at 37 ºC in 5% CO2 for 24 h. After 24
h, an additional 100 μL of complete medium with either: no
additions (negative control), 0.1% DMSO (solvent control), 10
μg/mL Taxol (positive control), or different concentrations of OSC6
ranging from 0.39 μM to 50 μM were added, and incubated for 72
h. Then, 20 μL of MTT solution (5 mg/mL) were added to the
culture medium, and incubated for 4 h. Next, the medium was
removed, and the formazan was dissolved with 150 μL DMSO.
Absorbance values were measured at 490 nm using an enzyme-
linked immunosorbent Reader (EL-x800, BioTek Instruments,
Winooski, VT, USA). The IC50 value was the concentration of OSC6
that resulted in 50% inhibition of cell growth, which was
graphically calculated as a comparison with control growth.
The tea seeds (2.0 kg) were crushed into powder and
extracted three times with 70% EtOH (3 × 10 L) at 60 ºC under
reflux for 4 h each time. The extract was subjected to reduced
pressure evaporation to obtain EtOH concentrated solution (350
g), which was suspended in H2O and extracted successively with
petroleum ether, EtOAc and n-BuOH. The n-BuOH fraction (80 g)
was subjected to CC on silica gel and eluted with a gradient of
EtOAc-MeOH (100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60,
30:70, 20:80, 10:90, 0:100, each 8.0 L). Each fraction was
collected, yielding eight major fractions (A - H), and analyzed by
TLC. Fraction G (6.5 g) was submitted to CC on Sephadex LH-20 in
MeOH to remove the pigments and flavones, yielding four major
fractions ( Ⅰ - Ⅳ ). Fraction Ⅱ (2.0 g) was subjected to
semipreparative HPLC [YMC-Pack ODS-A, CH3CN-0.5% aqueous
HCOOH (40:60, v/v), 2 mL/min] to yield five fractions [Fr. 1 (0.83
g), Fr .2 (0.12 g), Fr. 3 (0.06 g), Fr. 4 (0.11 g), and Fr. 5 (0.55 g)].
Fraction 4 was further purified by HPLC [Agilent C18, CH3CN-0.5%
aqueous HCOOH (44:56, v/v), 1 mL/min] to afford oleiferasaponin
C6 (6.7 mg).
Cell cycle analysis
HL-60 and BEL-7402 cells were seeded in 6-well plates at
5×105 cells/well, and incubated for 24 h until adherent. Next, HL-
60 and BEL-7402 cells were treated with various concentrations of
OSC6 for 72 h, respectively. After treatment with OSC6, the cells
were harvested, washed, suspended in ice-cold PBS, and fixed in
70% ethanol at 4 ºC overnight. The cells were stained for total
DNA content with RNase A and propidium iodide (PI) staining
buffer according to the manufacturer’s instructions (Becton
Dickinson). Cell cycle distribution was analyzed using a flow
cytometer (Becton Dickinson, San Jose, CA, USA) and ModFit
software V3.0 (Verity Software House, Topsham, ME, USA).
OSC6: White amorphous powder; UV (MeOH) λmax nm (log
λ
): 208 (3.98), 254 (4.17), 279 (4.32); IR (KBr) νmax (cm-1): 3416,
1
2950, 2927, 1716, 1638, 1450, 1384, 1309, 1164, 1078, 1046; H
NMR (pyridine-d5, 600MHz) and 13C NMR (pyridine-d5, 150MHz)
spectroscopic data, was shown in Table 1, Fig. S3, S4; HR-ESI-MS
(positive ion mode): m/z 1345.5824 [M + Na]+ (calcd for C65H94O28
Na, 1345.5829), the data were shown in Fig. S2.
Acid hydrolysis and GC-MS analysis of the sugar moieties in OSC6
The configuration of sugar units was established after
hydrolysis of OSC6 with 1 M HCl, pyridine containing L-cysteine
methyl ester hydrochloride, trimethylsilylimidazole and
determination of the retention times by GC-MS operating under
the experimental conditions previously reported by Zong et al.10
The standard sugar samples were subjected to the same reaction
and GC-MS conditions. The sugar units of OSC6 were identified by
comparison with authentic samples: D-xylose (tR 16.72 min), D-
galactose (tR 22.30 min), D-glucuronic acid methyl ester (tR 23.34
min). D-xylose, D-galactose and D-glucuronic acid methyl ester
were identified in a ratio of 1:2:1 for OSC6.
Cell apoptosis analysis
Cell apoptosis was assessed using the Annexin V-APC/7-AAD
double-staining apoptosis detection kit (Becton Dickinson, San
Jose, CA, USA). In brief, HL-60 and BEL-7402 cells were treated
with DMSO or OSC6 for 24 h. Next, cells were washed with ice-cold
PBS, and incubated for 15 min at room temperature in the dark.
The cells were collected and stained according to the
manufacturer’s instructions. The apoptosis data acquisition and
analysis was performed by a FACS Calibur flow cytometer. Basal
apoptosis were determined on negative control cells.
Cell culture
Western blot analysis
Human hepatocellular carcinoma BEL-7402, human gastric
carcinoma BGC-823, human breast cancer MCF-7, human
promyelocytic leukemia HL-60 and human oral epidermoid
carcinoma KB cell lines were obtained from KeyGEN BioTECH
(Nanjing, China). Cells were cultured in RPMI-1640 complete
medium supplemented with 10% fetal bovine serum, 1%
penicillin-streptomycin (100 U/mL penicillin and 100 μg/mL
streptomycin) and 2 mM L-glutamine at 37 ºC in a 5% CO2
humidified atmosphere.
HL-60 and BEL-7402 cells (2×105 cells/well) were seeded in 6-
well plates. After 24 h, the medium was replaced with fresh
culture medium containing different concentrations of OSC6 or
DMSO for 72 h. Then cultured cells were lysed in ice-cold RIPA
buffer supplemented with protease and phosphatase inhibitors
(Pierce, Rockford, IL, USA). Samples were maintained on ice for 30
min and then centrifuged at 13000 x g for 10 min, and the
supernatants were collected and quantified. Total protein
concentration was quantified using BCA protein assay. Equal
amount of samples were subjected to 12% SDS-PAGE, transferred
Cell viability assay
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2016, 00, 1-3 | 3
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Zong, Jianfa
