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resonance (NMR) spectrometry, gas chromatography (GC), peri-
odic acid oxidation, Smith degradation and methylation combined
with GC–mass spectrometry (GC–MS), and to investigate the anti-
tumor activities in vitro of purified fractions by determining their
inhibitory rates against human gastric cancer BGC-823 cells.
2.3.2. Monosaccharide composition analysis
The monosaccharide composition was analyzed according to
the method reported (Qiao et al., 2010) with minor changes.
Briefly, polysaccharides sample (5 mg) was hydrolyzed with 2 mL
of 2 M trifluoroacetic acid (TFA) at 120 ◦C for 2 h. The hydrolyzates
were repeatedly co-distilled with methanol to dryness and con-
verted into their corresponding aldonitrile acetates by the addition
of hydroxylammonium chloride (10 mg), inositol (5 mg), pyridine
(0.6 mL) and acetic anhydride (1.0 mL). The aldonitrile acetate
derivatives of standard monosaccharides were prepared in the
same way. Then, all the derivatives were analyzed by GC (GC 6890N,
Agilent) equipped with a flame ionization detector (FID) and an HP-
5 fused silica capillary column (30 m × 0.32 mm × 0.25 mm). The
operation conditions of GC were as following: flow rates of N2, H2
and air were 25, 30 and 400 mL/min, respectively; the temperature
of injector and detector were set at 250 and 280 ◦C, respectively;
programed at a rate of 3 ◦C/min to 210 ◦C and held for 4 min.
2. Materials and methods
2.1. Materials
The dried bulbs of A. macrostemon Bunge were obtained from
Anhui Huilong Co., Ltd (Hefei, China). Arabinose, rhamnose, fucose,
xylose, galactose, glucose, mannose, dimethyl sulfoxide (DMSO),
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT), DEAE-52 cellulose and Sephadex G-100 were purchased
from Sigma Chemical Co. (St. Louis, MO, USA). Human gastric can-
cer BGC-823 cell line was obtained from the Cell Bank of Shanghai
Institute of Cell Biology (Shanghai, China). RPMI-1640 media was
purchased from Gibco/Invitrogen (Grand Island, NY, USA). All other
chemicals used in the experiment were of analytical grade.
2.3.3. Molecular weight determination
Homogeneity and the molecular weights of fractions were ana-
lyzed according to the method reported (Zhang, Gu, et al., 2010)
with slight modification. A HPLC apparatus (Agilent 1100) equipped
with a TSK-Gel G3000 SWXL column (7.8 mm × 300 mm, Tosoh
Corp., Tokyo, Japan) and a refractive index detector (RID). Sample
(20 L) was injected in and eluted with 0.1 M Na2SO4 (dissolved in
temperature of the column and detector was set at 25 ◦C. The cali-
bration curve for molecular weight determination was made using
a series of Pullulan P-800, P-400, P-200, P-100, P-20, P-10 and P-
5 (Shodex standard P-82, Showadenko, Tokyo, Japan) as standards,
following the method described by Alsop and Vlachogiannis (1982).
2.2.1. Preparation of AMP40
The preparation of AMP40 was carried out according to our
method reported (Zhang et al., 2012). Briefly, the pretreated bulbs
were extracted with water (the ratio of water to raw material
12 mL/g) three times at 87 ◦C for 100 min. After centrifugation
(4500g, 10 min), the supernatants were concentrated under vac-
uum and treated with alcohol to a concentration of 40% (v/v)
for precipitation at 20 ◦C overnight. The precipitate was dissolved
in water and dialyzed against distilled water (exclusion limit
8–14 kDa). The non-dialyzable portion was frozen at −20 ◦C and
freeze-dried to afford AMP40.
2.3.4. FT-IR analysis
The FT-IR spectra of polysaccharides were recorded with a Nico-
let 200 FT-IR spectrometer (Thermo Fisher Scientific Inc., Waltham,
(KBr) pellet method (Kumar, Joo, Choi, Koo, & Chang, 2004).
2.2.2. Purification of AMP40
AMP40 was purified by column chromatography. Firstly, AMP40
(200 mg) was dissolved in 5 mL of distilled water and applied to a
water, 0.1, 0.3 and 0.5 M NaCl solutions at a flow rate of 1.0 mL/min.
Fractions (10 mL/tube) were collected by a fraction collector and
analyzed for the carbohydrate content by the phenol-sulfuric acid
assay (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). The result-
ing fractions were further purified by a column of Sephadex G-100
(1.5 × 70 cm), eluting with 0.1 M NaCl solution at a flow rate of
0.05 mL/min. Fractions were analyzed for the carbohydrate con-
tent, collected, concentrated, dialyzed against distilled water and
lyophilized, affording the neutral and acidic polysaccharide frac-
tions of AMP40N and AMP40S.
2.3.5. Periodic acid oxidation, Smith degradation and GC analysis
Assay of periodic acid oxidation, Smith degradation and GC were
performed by using the method reported by Qiao et al. (2010) with
slight modification. Briefly, sodium metaperiodate (NaIO4, 0.015 M)
and sodium iodate (NaIO3, 0.015 M) were mixed at different ratios
and the mixed solution was detected on spectrophotometer at
223 nm, affording a standard curve of NaIO4. Polysaccharide sam-
ple (25 mg) was dissolved in 25 ml 15 mmol/L of NaIO4. The solution
was kept in the dark at 4 ◦C with interval stirring, 0.1 mL aliquots
were withdrawn at 12 h intervals, diluted to 25 mL with distilled
water, and absorbance at 223 nm of dilution was detected. When
absorbance did not descend, the reaction was complete and the
excess of NaIO4 was decomposed with ethylene glycol (1.0 mL).
Consumption of NaIO4 was calculated according to the NaIO4
standard curve. Yield of formic acid (HCOOH) produced was deter-
mined by titration and calculated according to the consumption
of NaOH. The reaction mixture was reduced with sodium borohy-
dride (NaBH4, 50 mg) for 20 h at 25 ◦C, and then was adjusted to pH
5.5–7.0 with acetic acid (0.1 M). The reaction solution was dialyzed,
2.3.1. Determination of contents of carbohydrate, protein, uronic
acid and sulfuric radical
The content of carbohydrate was determined by the phenol-
sured by a modified hydroxydiphenyl assay (Blumenkrantz &
Asboe-Hansen, 1973) with d-glucuronic acid as the standard. The
content of sulfuric radical was determined according to the method
reported (Dodgson & Price, 1962).
2.3.6. Methylation and GC–MS analysis
Polysaccharide sample was methylated three times using the
method of Ciucanu and Kerek (1984) with slight modifications. The
completeness of methylation was confirmed by the disappearance