International Journal of Biological Macromolecules 51 (2012) 259–265

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Characterization and in vitro antioxidant activities of polysaccharides from

Pleurotus ostreatus
Yunxia Zhang, Ling Dai ∗ , Xiaowei Kong, Liangwen Chen
School of Life Science, Anhui University, Hefei 230039, Anhui, China

a r t i c l e i n f o a b s t r a c t

Article history: Two polysaccharide fractions (PSPO-1a and PSPO-4a) were isolated from the fruiting bodies of
Received 20 March 2012 Pleurotus ostreatus using ethanol precipitation, anion-exchange chromatography and gel permeation
Received in revised form 18 April 2012 chromatography. Both fractions were heteropolysaccharide containing protein and uronic acid. PSPO-
Accepted 3 May 2012
1a was composed of mannose, glucose, galactose, xylose and rhamnose with a molar ratio of
Available online 10 May 2012
2.47:0.91:1.00:1.66:3.87. PSPO-4a was composed of only three monosaccharides: rhamnose, mannose
and galactose with a molar ratio of 0.92:2.69:1.00. The average molecular weight of PSPO-1a and PSPO-4a
Keywords:
determined by HPLC were estimated to be 1.8 × 104 Da and 1.1 × 106 Da respectively. The in vitro tests
Pleurotus ostreatus
Polysaccharide
revealed that two polysaccharides were natural potential antioxidant. Both polysaccharides presented
Characterization stronger DPPH radical and superoxide anion radical scavenging activity with increasing concentrations,
Antioxidant activities but less effective on scavenging hydroxyl radical. Compared with PSPO-4a, PSPO-1a was the more effec-
tive free-radical scavenger. In conclusion, the two polysaccharides may be useful as a naturally potential
antioxidant agent for application in food and medicinal fields.
© 2012 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
2. Materials and methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
2.1. Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
2.2. Preparation of polysaccharides extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
2.3. Fractionation and purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
2.4. Spectra analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
2.5. Homogeneity and molecular weight determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
2.6. Chemical and monosaccharide composition analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
2.7. Antioxidant assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
2.7.1. DPPH radical-scavenging assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
2.7.2. Superoxide anion radical assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
2.7.3. Hydroxyl radical assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
2.8. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
3.1. Isolation and purification of polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
3.2. Spectroscopic characteristics of the polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
3.3. Homogeneity and average molecular weight of PSPO-1a and PSPO-4a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262
3.4. Chemical and monosaccharide composition of the polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262
3.5. Results of free radical scavenging assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262
4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265

∗ Corresponding author. Tel.: +86 13355606281.

E-mail address: dailing@ahu.edu.cn (L. Dai).

0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijbiomac.2012.05.003
260 Y. Zhang et al. / International Journal of Biological Macromolecules 51 (2012) 259–265
1. Introduction
Mushrooms have been part of the human diet for thousands of
years in many Asian countries like China, Korea and Japan. It is well
known that mushrooms are valuable health food since they are low
in calories, fats and rich in proteins, vitamins and rare minerals.
Nowadays, medicinal beneficial effects of mushrooms are becom-
ing increasingly recognized and have attracted much attention in
their application in food and pharmaceuticals [1,2].
Pleurotus ostreatus is a popular and important edible mush-
rooms cultivated commercially worldwide. It has been reported
that dietary supplements of P. ostreatus had hypocholesterolemic
effects in animal models and diabetic patients [3,4]. It was also
shown that the powder of P. ostreatus had potent antinociceptive
effect in rats which fed with the powder [5]. In addition, a num-
ber of studies demonstrated that polysaccharide isolated from P.
ostreatus had many medicinal activities. These activities included
inhibition of tumor cell proliferation both in vivo and in vitro,
modulation of immune activity by elevation NK (natural killer)
cell cytotoxicity and macrophage production of nitric oxide [6–8].
Antioxidant activities and anti-inflammatory activities were also
reported. Studies conducted in animal models demonstrated that
treatment with polysaccharides from P. ostreatus could protect rat
hepatocytes from oxidative damage induced by CCl4 , decrease inci-
dence of precancerous lesions in rat colon and decrease activities
of proinflammatory cytokines TNF-␣, IL-1 and IL-6 in the serum of
arthritic rats [9–12]. More and more data show that polysaccha-
rides from P. ostreatus have important bioactivities, which make
them worth researching in depth.
In the present study, we investigated two polysaccharides iso-
lated from the fruit body of P. ostreatus. The work was undertaken
to characterize partial structural features and assess antioxidant
activity in vitro.
2. Materials and methods
2.1. Materials
The fruit bodies of P. ostreatus (Jacq.:Fr.) Quel. were obtained
from Field Company (Anhui, China) and dried at 60 ◦ C for use.
DEAE-cellulose 52 was purchased from Whatman (USA), superdex-
75HR column was purchased from Amersham Biosciences. The
monosaccharide standards, trifluoroacetic acid (TFA) and 2, 2-
diphenyl-1-picrylhydrazy (DPPH) were purchased from Sigma.
Dextran standards T-10, T-40, T-70, T-100, T-500, T-2000 were
purchased from Pharmacia (Sweden). All other reagents were of
analytical pure grade, purchased from Shanghai Chemical Reagent
(China).
2.2. Preparation of polysaccharides extract
100 gram of dry powder from fruiting bodies of P. ostreatus was
extracted with hot water (60 ◦ C, 3 h, 1:30, w/w). Extract was filtered
and concentrated at 60 ◦ C in a rotary evaporator under reduced
pressure, precipitated by 95% ethanol in 1:3 ratio (extract:ethanol,
v/v) and kept overnight at 4 ◦ C, then centrifuged (5000 × g, 20 min).
Resulted precipitate was freeze dried, and crude polysaccharide
PSPO was obtained.
2.3. Fractionation and purification
Crude polysaccharide was fractionated and purified by anion-
exchange chromatography. The crude polysaccharide (500 mg)
was dissolved in 10 mL distilled water, and loaded on the DEAE-
cellulose 52 column (2.6 cm × 40 cm). The column was eluted with
distilled water, followed by a 0–1 mol/L linear gradient of NaCl
at a flow rate of 4 mL/6 min. The elution of polysaccharide was
monitored using phenol–sulfuric acid assay. The four fractions con-
taining polysaccharide were pooled, dialyzed and then freeze dried
respectively. Two major fractions were further purified by gel per-
meation chromatography on a superdex-75HR column performed
on AKTA basic system (Amersham Biosciences), with distilled water
as eluent at a flow rate of 0.5 mL/min. Two homogeneous polysac-
charides fractions were obtained respectively and lyophilized for
further investigation.
2.4. Spectra analysis
Fourier-transform infrared (FT-IR) spectra were recorded from
the samples in KBr pellet on a FT-IR spectrophotometer (NEXUS-
870, Nicolet Instrument Co., USA). UV–vis spectroscopy analyses
were conducted on Ultraviolet–Visible–Near-Infrared spectropho-
tometer (U-4100, Hitachi Ltd., Japan). The polysaccharide solution
was prepared by suspending the sample in distilled water to a
concentration of 1.0 mg/mL for UV–vis measurement in the wave-
length range of 190–700 nm.
2.5. Homogeneity and molecular weight determination
Determination of molecular weight was done according to the
reported method of HPLC with slight modification [13]. Measure-
ments were performed on a Waters 515 high-performance liquid
chromatography (HPLC) instrument (Waters, USA) with TSK-GEL
G4000PWxl gel permeation column (˚ 7.5 mm × 300 mm, Tosoh
Corp., Japan), and detected with Waters 410 refractive index
detector (RID). The operation was performed under the follow-
ing conditions: concentration of polysaccharide, 2.5 mg/mL; mobile
phase, 0.7% Na2 SO4 with a flow rate of 1.0 mL/min; column temper-
ature, 30 ◦ C; injection volume, 10 ␮L. The column was calibrated
with Dextran T-series standard of known molecular weight (T-
10, T-40, T-70, T-100, T-500 and T-2000). The average molecular
weight of polysaccharide was calculated by reference to the cali-
bration curve made by Dextran T-series standards.
2.6. Chemical and monosaccharide composition analysis
The polysaccharide content was examined using
phenol–sulfuric acid colorimetric method with glucose as a
standard [14]. The protein content was determined by the Brad-
ford method using bovine serum albumin (BSA) as a standard
[15]. Uronic acid content was determined by m-hydroxyl diphenyl
method [16] using glucuronic acid as the standard.
The hydrolysis of polysaccharide was conducted using the
method described by Carnachan and Harris [17]. 20 mg of PSPO-1a
and PSPO-4a were respectively hydrolyzed with 2 mL of triflu-
oroacetic acid solution (TFA, 2 mol/L) at 100 ◦ C for 6 h, and the
residual TFA was removed by methanol. Then the hydrolysates of
PSPOs were analyzed by HPLC/ELSD system (Waters 2420 ELSD).
The column used was ZORBAX carbohydrate analysis column (˚
250 mm × 4.6 mm, Agilent, USA). The column was eluted with the
mixture of acetonitrile and distilled water (4:1, v/v) at a flow
rate of 1 mL/min. A monosaccharide standard mixture, including
d-glucose (Glc), d-galactose (Gal), l-arabinose (Ara), l-rhamnose
(Rha), d-mannose (Man) and d-xylose (Xyl) was used for monosac-
charide composition identification with the same method.
2.7. Antioxidant assays
2.7.1. DPPH radical-scavenging assay
The DPPH radical scavenging activity of polysaccharides was
assayed according to the method of Shimada et al. [18]. Briefly, 2 mL
of polysaccharide solution in the different concentration range was
 Y. Zhang et al. / International Journal of Biological Macromolecules 51 (2012) 259–265 261

added to 2 mL of DPPH solution (0.2 mmol/L, in 95% ethanol solu-

tion). The mixture was shaken and reacted for 30 min at 25 ◦ C in the
dark. The blank control was distilled water instead of polysaccha-
ride. The absorbances of the mixtures were read at 517 nm against
a blank. The radical scavenging activity was measured as a decrease
in the absorbance of DPPH and was calculated using the following
equation:
 A1 − A2

Scavenging rate (%) = 1 − × 100
A0
where A0 is the absorbance of the control group (water instead of
test sample solution), A1 is the absorbance of the test group and A2
is the absorbance of the samples only (95% ethanol instead of DPPH
solution).

2.7.2. Superoxide anion radical assay

The scavenging effect of the samples towards superoxide anion
radicals was investigated by the method of Marklund and Mark-
lund [19] with a little modification. Briefly, the reaction mixture
that contained 2 mL polysaccharide solution of different concen-
tration and Tris–HCl (50 mmol/L, pH 8.2, 5 mL) was incubated at Fig. 1. The crude polysaccharide PSPO was applied to a column of DEAE-cellulose
52 anion-exchange chromatography. The column was eluted with distilled water,
25 ◦ C for 20 min. Then, 0.4 mL pyrogallol solution (3 mmol/L, 25 ◦ C)
followed by linear gradient of sodium chloride solution. Detection was by measur-
was added rapidly. The reaction was terminated after 4 min by ing the absorbance at 490 nm with the phenol–sulfuric acid method. The fractions
adding 0.5 mL HCl, the absorbance was measured at 320 nm after containing the polysaccharides were pooled and named as PSPO-1, PSPO-2, PSPO-3
10 min. The scavenging activity of superoxide radicals was calcu- and PSPO-4, respectively.
lated according to the following equation:
 A1 − A2

Scavenging rate (%) = 1 − × 100
A0 yield of PSPO was 5.76% (w/w) of the dried material. Crude PSPO
was then chromatographed on a DEAE-cellulose 52 column. PSPO-1
where A0 is the absorbance of the control group (Tris–HCl instead
and PSPO-2 were acquired from distilled water eluate, and PSPO-
of test sample solution), A1 is the absorbance of the test group and
3 and PSPO-4 were obtained from the NaCl eluate (Fig. 1). Next,
A2 is the absorbance of the samples only (0.1 mol/L HCl instead of
the fractions PSPO-1 and PSPO-4 were subjected to Superdex-75HR
pyrogallol solution).
column with an AKTA basic system for further purification, yield-
ing two homogenous polysaccharide fractions called PSPO-1a and
2.7.3. Hydroxyl radical assay
PSPO-4a respectively.
Assessment of the scavenging ability of PSPOs on hydroxyl rad-
icals was performed according to the method previously described
3.2. Spectroscopic characteristics of the polysaccharides
by Smirnoff and Cumbes [20] with a minor modification. Briefly,
2 mL different concentration of polysaccharide solution was mixed
Fourier transforms infrared spectra profiles of the two PSPOs
with 1 mL FeSO4 (9 mmol/L) and 2 mL salicylic acid–ethanol solu-
were found to be similar, as shown in Fig. 2. A feature of polysac-
tion (9 mmol/L), then added 2 mL H2 O2 (8.8 mmol/L), mixtures
charide absorption peak at the region of proximately 3400 cm−1
were incubated for 60 min at 25 ◦ C. The distilled water was used as
the blank control. The absorbance of the mixtures was measured
at 510 nm. Hydroxyl radical scavenging activity was calculated by
the following equation:
 A1 − A2

Scavenging rate (%) = 1 − × 100
A0
where A0 is the absorbance of the control group (water instead of
test sample solution). A1 is the absorbance of the test group and
A2 is the absorbance of the samples only (water instead of H2 O2
solution).

2.8. Statistical analysis

Results were expressed as mean ± standard deviation of trip-

licate analysis. Statistical comparisons were performed using the
Student’s t test. Differences were considered statistically significant
at p < 0.05.

3. Results

3.1. Isolation and purification of polysaccharides

Under the optimal extraction conditions (1:30, w/w, 60 ◦ C, 3 h),

crude polysaccharide of P. ostreatus (PSPO) was prepared. The total Fig. 2. The FT-IR spectra of PSPO-1a and PSPO-4a.
262 Y. Zhang et al. / International Journal of Biological Macromolecules 51 (2012) 259–265
Fig. 3. The UV–vis spectra of PSPO-1a and PSPO-4a in the wavelength range of
190–700 nm.
was the stretching vibration of O H bond, the other feature of
polysaccharide absorption peak was the stretching vibration of
C H at region around 2928 cm−1 . Two prominent absorption bands
at 1071 cm−1 , 1057 cm−1 in the range of 1200–1000 cm−1 sug-
gested that the monosaccharide of PSPOs had a pyranose ring [21],
an stretching peak appeared at 1633 cm−1 and a weak stretching
peak at 1418 cm−1 , 1350 cm−1 , suggesting the presence of carboxyl
groups. Furthermore, a small absorption at about 890 cm−1 could be
associated with ␤-glycoside linkages between the sugar units. It can
be concluded that PSPOs belong to ␤-type hetero polysaccharide
with pyran group.
The UV spectra of fractions (PSPO-1a and PSPO-4a) were pre-
sented in Fig. 3. The largest absorbance peaks of PSPO-1a and
PSPO-4a were 195 nm and 200 nm, respectively, which revealed
characteristics of polysaccharide. There was weak absorption at
280 nm in PSPO-1a, indicating very small amounts of proteins con-
tained.
3.3. Homogeneity and average molecular weight of PSPO-1a and
PSPO-4a
PSPO-1a and PSPO-4a obtained from anion exchange chro-
matography and gel permeation chromatography were detected by
HPLC for their homogeneity and molecular weight. The HPLC profile
demonstrated that both PSPO-1a and PSPO-4a had a symmetrical
sharp peak revealing that PSPO-1a and PSPO-4a were homoge-
neous fractions (Fig. 4). The average molecular weight of PSPO-1a
and PSPO-4a were calculated by HPLC. The equation of the stan-
dard curve was: log Mw = 8.8096–0.4712t, R2 = 0.9904 (where Mw
is molecular weight, and t is retention time). The average molecular
weight of PSPO-1a and PSPO-4a were estimated to be 1.8 × 104 Da
and 1.1 × 106 Da respectively by reference to the dextran standards
calibration curve.
3.4. Chemical and monosaccharide composition of the
polysaccharides
The content of protein, total sugar, uronic acid contents and
monosaccharide composition in PSPOs were analyzed and sum-
marized in Tables 1 and 2. The total sugar content of PSPO-1a
Table 1
Chemical composition of PSPO-1a and PSPO-4a (wt%).
Components Carbohydrate Protein Uronic acid
PSPO-1a 71 1.23 5.61
PSPO-4a 31.66 0.84 7.91
and PSPO-4a was determined to be 71% and 32% with a pro-
tein content of 1.23% and 0.84% respectively. The uronic acid
content evaluated in PSPO-1a and PSPO-4a was 5.61% and 7.91%
respectively.
Monosaccharide composition of PSPOs was analyzed by
HPLC/ELSD and shown in Fig. 5b and c. Compared with
the monosaccharide standards shown in Fig. 5a, PSPO-1a
was composed of Rha, Xyl, Man, Glc, and Gal in a molar
ratio of 3.87:1.66:2.47:0.91:1.00, PSPO-4a was composed of
only three monosaccharides: Rha, Man and Gal in a molar
ratio of 0.92:2.69:1.00. From the results given above, we
can find that the two fractions have different chemical
and monosaccharide composition, so it was more meaningful
to investigate the biological activity of the two fractions.
3.5. Results of free radical scavenging assay
The scavenging effects of PSPO-1a and PSPO-4a polysaccharides
on three different radicals were shown in Fig. 6a–c. The results
showed that PSPOs were potential free-radical scavengers, and that
their activities against the radicals were closely associated with
polysaccharide compositions.
Most research showed that the DPPH scavenging activities of
antioxidants depended on their hydrogen donating abilities [22].
Fig. 6a demonstrated DPPH scavenging activity, expressed in per-
cent, caused by different concentrations of PSPO-1a and PSPO-4a.
Scavenging activities of PSPO-1a and PSPO-4a against the DPPH
radical were 62.5 ± 1.03% and 54.9 ± 0.8% at 2.1 mg/mL, respec-
tively. In addition, PSPO-1a showed a better scavenging ability (IC50
1.43 ± 0.055 mg/mL) than PSPO-4a (IC50 2.3 ± 0.067 mg/mL). These
results showed that PSPO-1a was a prospective DPPH scavenging
agent.
Superoxide radical could produce a colored compound due to
the color change from purple to yellow, the absorbance at 320 nm
increased when the superoxide anion radical was scavenged by
antioxidant [23]. The superoxide anion radicals scavenging activi-
ties of PSPOs were determined in this study. As shown in Fig. 6b,
the effects of PSPO-1a and PSPO-4a on scavenging superoxide
anion radicals were in a concentration-dependent manner. The
IC50 values of PSPO-1a and PSPO-4a were 2.0 ± 0.066 mg/mL and
4.8 ± 0.067 mg/mL respectively. Also we can find PSPO-1a exhibited
a higher effect than PSPO-4a.
Salicylic acid has the ability to absorb • OH to bring coloring
material. Added hydroxyl radical scavengers compete with sali-
cylic acid, which makes the coloring material down. The method
could be used to evaluate the hydroxyl radical scavenging abil-
ity of polysaccharide [24]. Scavenging hydroxyl radical effects
were observed in PSPO-1a and PSPO-4a polysaccharides. The
results showed that the two polysaccharides had weak activities
on the scavenging effects of hydroxyl radicals as compared with
Table 2
Monosaccharide compositions of PSPO-1a and PSPO-4a (mol%).
Components Rha Xyl Man Glc Gal
PSPO-1a 3.87 1.66 2.47 0.91 1.00
PSPO-4a 0.92 nd 2.69 nd 1.00
nd = not detected.
 Y. Zhang et al. / International Journal of Biological Macromolecules 51 (2012) 259–265
Fig. 4. The high-performance liquid chromatogram of polysaccharide PSPOs from Pleurotus ostreatus. (a) PSPO-1a and (b) PSPO-4a.
scavenging effects of DPPH and superoxide radicals. The scav-
enging activity only increased to 22.0% and 17.0% at 3.0 mg/mL,
respectively (Fig. 6c).
4. Discussion
Polysaccharides obtaining from P. ostreatus mycelia, fruit body
and fermented broth have been fractionated and characterized.
The most reported polysaccharides from P. ostreatus were glu-
cans with different types of glycosidic linkages, such as branched
␤-glucans and ␣-glucans [25–29]. However, other kinds of bio-
logically active glycans of different structures have also been
described. Sun and Liu [30] reported a water-soluble polysac-
charide (POP) which mainly composed of ␣-d-galactopyranosyl
residues, ␤-d-glucose residue only terminated at the O-2 posi-
tion of every other galactosyl residue. Immunological activity assay
showed that POP could increase the ConA (Concanavalin A) or LPS-
induced lymphocytes proliferation. In our research, we obtained
two water-soluble polysaccharides with potent antioxidant activ-
ity from the fruiting body of P. ostreatus. PSPO-1a contained five
kinds of monosaccharides, mainly composed of Rha, Man and Xyl.
PSPO-4a contained three kinds of monosaccharides, Man was the
263
major monosaccharide. Monosaccharide composition of these two
PSPOs was different from previous reported polysaccharides which
contained predominantly glucose. Based on the results above, it
was reasonable to conclude that PSPOs were novel complicated
heteroglycans. In addition, many studies have shown that water-
soluble heteroglycans isolated from mushroom have a variety of
biological activities.
It is well-known that excessive reactive oxygen species (ROS),
such as singlet oxygen, hydroxyl radicals, superoxide anion rad-
ical and hydrogen peroxide, are capable of causing damage to
biological macromolecules, therefore, ROS are related to the patho-
genesis of various human diseases including cancer, aging, and
many chronic diseases. Natural antioxidants play a key role in
health maintenance and prevention of the chronic and degener-
ative diseases. Polysaccharides from plants and fungi have been
recognized to be a promising group of antioxidant compounds.
Many studies have shown that polysaccharides improve the activ-
ity of antioxidant enzymes in the body, scavenge free radicals,
and inhibit lipid oxidation [31,32]. In the present work, several
in vitro assays were applied to evaluate the antioxidant poten-
tial of polysaccharides. Our findings indicated that PSPO-1a and
PSPO-4a possessed potent antioxidant activities. Between the
264 Y. Zhang et al. / International Journal of Biological Macromolecules 51 (2012) 259–265

Fig. 5. HPLC/ESLD analysis of monosaccharide composition of polysaccharide PSPOs

from Pleurotus ostreatus. (a) A mixture of standard monosaccharides, (b) the
monosaccharide composition of PSPO-1a, and (c) the monosaccharide composition
of PSPO-4a.

two polysaccharides, PSPO-1a appeared to have higher antioxi-

dant activity, suggesting that the composition and the ratio of
monosaccharides, as well as the contents of uronic acid could
Fig. 6. Effect of polysaccharide PSPO-1a and PSPO-4a on the free-radical scavenging.
affect their antioxidant activity. It has been reported that the basic (a) The effect on DPPH radical, (b) the effect on superoxide anion radical, and (c) the
structure or molecular weight of polysaccharides may play an effect on hydroxyl radical. Values are means ± SD (n = 3).
important role in influencing the bioactivities [33,34]. There-
fore, we speculate that the basic structure of polysaccharides
might be the key factor, but this needs to be further stud-
ied.
 Y. Zhang et al. / International Journal of Biological Macromolecules 51 (2012) 259–265
5. Conclusion
Polysaccharides such as ␤-d-glucans, heteropolysaccharides
and glycoprotein in mushrooms comprise one of the major sources
of bioactive compounds. Our results showed that PSPO-1a and
PSPO-4a, the novel water-soluble heteropolysaccharides isolated
from P. ostreatus, possess potent antioxidant activities, it suggests
that these polysaccharides may be useful as a naturally potential
antioxidant agent for application in food and medicinal fields. Fur-
ther work is now in progress with the aim to elucidation of fine
structure of polysaccharides such as the linkage of sugar chain,
branches and evaluates the possible antioxidant activity in vivo.
Acknowledgments
This study was supported by the 211 Project of Anhui University.
The authors are deeply grateful to Prof. Yeshou Shen for identifica-
tion of Pleurotus ostreatus.
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