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Y. Huang et al. / Carbohydrate Polymers 134 (2015) 752–760
2.6. Monosaccharide composition analysis
2.10. Nuclear magnetic resonance (NMR) analysis
The identification and quantification of the monosaccharides
of GCPB-2 were achieved by GC analysis. GCPB-2 (5 mg) was
hydrolyzed with 2 M trifluoroacetic acid (TFA) at 120 ◦C for 6 h in a
sealed glass tube. Then, the sample was neutralized with methanol.
The supernatant was added to 10mg hydroxylamine hydrochloride
and 1mL pyridine, and the mixture was incubated in a water bath
at 90 ◦C for 30 min. Then, acetic anhydride (1 mL) was added with
continuous heating. The hydrolysis product was converted into its
acetylated derivative and analyzed by the Agilent 6820 GC system
(Agilent, USA) with an OV-17 capillary column. The temperature
program of the column was set as follows: initial temperature of
200 ◦C was increased to 220 ◦C at the rate of 15 ◦C/min and held for
10 min. Subsequently, the temperature was increased from 220 ◦C
to 240 ◦C at 10 ◦C/min and maintained for 10 min. The injection
temperature was 250 ◦C.
The freeze-dried GCPB-2 (50 mg) was put in a 5mm NMR tube
and dissolved in 0.5 mL of D2O. All spectra were recorded at 27 ◦C
on a Bruker AV-500 spectrometer (Germany) operating at 500 MHz
for 1H NMR and 125 MHz for 13C NMR. Two-dimensional spectra
(HMQC and HMBC) were recorded by standard Bruker procedures.
2.11. Determination of DPPH• radical-scavenging activity
The free radical-scavenging activity of GCPB-2 was measured
by the 1, 1-diphenyl-2-picryl-hydrazyl (DPPH•) test, according to
the method of Wang, Gao, Zhou, Cai, and Yao (2008), with some
modifications. DPPH• ethanol solution (2 mL, freshly prepared at a
concentration of 0.1 mM) was added to 2 mL of aqueous solution of
GCPB-2 at gradient concentrations. The absorbance of the mixture
was measured at 517 nm after 30 min of incubation at 37 ◦C in the
dark. Ascorbic acid (Vc) was used as a positive control and distilled
water as the blank control. The lower absorbance of the reaction
mixture indicated the higher free radical-scavenging activity. The
capability to scavenge the DPPH• radical was calculated using the
following equation: Scavenging rate (%) = (1 − AS/Ai) × 100%, where
AS is the absorbance obtained for a sample (GCPB-2 or Vc) and Ai is
the absorbance of the blank.
2.7. Partial acid hydrolysis
GCPB-2 (40 mg) was hydrolyzed with 0.05 M TFA (25 mL) for 2 h
at 100 ◦C. The hydrolysate was dialyzed (Mw cutoff: 3000 Da) with
distilled water for 48 h. The outside solution of the dialysis bag was
collected and named GCPB-2a for GC analysis. Then, three times
of the volume of the inside solution of ethanol were added to the
inside solution, and the supernatant and precipitate fractions were
named GCPB-2b and GCPB-2c, respectively. These three fractions
were dried prior to GC analysis as mentioned in Section 2.6.
2.12. Determination of hydroxyl radical-scavenging activity
The scavenging activity for hydroxyl radicals was measured by
the Fenton reaction (Yu et al., 2007). Reaction was started by adding
H2O2 and incubating at 37 ◦C for 60 min. The absorbance of the
mixture at 536 nm was measured. The hydroxyl radical-scavenging
activity was calculated according to the following equation:
Scavenging ability (%) = [(A2 − A1)/(A0 − A1)] × 100%, where A0 is
the absorbance of the control (blank, without H2O2), A1 is the
absorbance in the absence of the sample, and A2 is the absorbance
in the presence of the sample.
2.8. Periodate oxidation and Smith degradation
GCPB-2 (30 mg) was oxidized with 0.015 M sodium periodate
(30 mL) and kept in the dark at 4 ◦C. Then the spectrophotometric
reading of the mixture was taken at 223 nm every 6 h using a
spectrophotometer until the optical density became stable. Then
2mL glycol was added to remove the excess sodium periodate and
determine the consumption of sodium periodate. The production
tap water and distilled water for 48 h and reduced with sodium
borohydride (80 mg) for 12 h at 25 ◦C. Then, the excess sodium
borohydride was neutralized by adding acetic acid (Rout, Mondal,
Chakraborty, & Islam, 2008). Subsequently, the solution was con-
centrated and subjected to complete hydrolysis with 2 M TFA. The
acid was removed by co-distillation with methanol under vacuum.
Finally, the products were acetylated and analyzed by GC.
3. Results and discussion
3.1. Isolation and purification of GCPB-2
The water-soluble polysaccharides named PB were isolated
from G. capense mycelia powder by derosination, sodium hydroxide
purification step by DEAE-52 cellulose chromatography, fractions
PB-1, PB-2, and PB-3 were eluted with 0.05 M, 0.1 M and 0.25 M
sodium chloride, respectively. Among them, PB-3 was already
reported in a previous study (Yi et al., 2015). PB-2 was then further
purified by DEAE Sepharose CL-6B and Sephadex G-75 gel-filtration
columns, successively. Only a sharp peak was detected in the DEAE
Sepharose CL-6B and Sephadex G-75 gel-filtration chromatogra-
phy. Then the sharp peak was collected as GCPB-2. The yield of
GCPB-2 from crude polysaccharide PB was about 15.2%.
2.9. Methylation analysis
GCPB-2 (10 mg) was methylated by the Ciucanu and Kerek
(Ciucanu
& Kerek, 1984) method. The complete methyla-
tion was examined by the disappearance of the OH band
(3200–3700 cm−1) in the IR spectrum. The methylated polysaccha-
ride was treated with 2 M TFA (2 mL) for 6 h at 120 ◦C in a sealed
tube and the hydrolysate was concentrated to dryness. Then the
methylated product was reduced with sodium borohydride and
then acetylated with the same amount of acetic anhydride and
pyridine for 1 h at 95 ◦C. Alditol acetate was analyzed by GC–MS
(GCMS-QP 2010, Shimadzu, Kyoto, Japan). The sample (1 L) was
injected with a split ratio of 50:1 and the ion source of the mass
spectrometer was set at 200 ◦C. The temperature program was set
as follows. The initial column temperature of 150 ◦C was increased
to 180 ◦C at the rate of 10 ◦C/min. The rate was changed to 15 ◦C/min
from 180 ◦C to 260 ◦C. Afterward, the temperature was maintained
for 5 min at 260 ◦C with an injection temperature of 220 ◦C.
3.2. Homogeneity and molecular weight determination of GCPB-2
The HPGPC analysis of GCPB-2 exhibited a single and symmetri-
cal peak, basically declaring the homogeneity of the polysaccharide.
Its molecular weight was determined as 1.03 × 105 Da. No absorp-
tion was detected at either 280 or 260 nm by UV spectrum,
demonstrating the absence of nucleic acids and protein. The optical
rotation of GCPB-2 was [˛]2D5 = + 161◦ (c 1.0, H2O).