Biological and physicochemical properties of two polysaccharides from the mycelia of Grifola umbellate

Article abstract of DOI:10.1016/j.carbpol.2013.03.002

In the present study, we firstly reported the antioxidant, antitumor and immunomodulatory effects of two polysaccharides (GUMP-1-1 and GUMP-1-2) isolated from Grifola umbellata mycelia. Chemical analysis indicated that two polysaccharide fractions contain

Full text of DOI:10.1016/j.carbpol.2013.03.002

Carbohydrate Polymers 95 (2013) 740–745
Contents lists available at SciVerse ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Biological and physicochemical properties of two polysaccharides
from the mycelia of Grifola umbellate
Yunpeng Bia,1, Ye Miaob,c,1, Yan Han , Jian Xu , Qing Wang
d
e
f,∗
a
Department of Prosthodontics, School of Stomatology, The Fourth Military Medical University, Xi’an 710032, PR China
Department of Oral Biology, School of Stomatology, The Fourth Military Medical University, Xi’an 710032, PR China
Oncology Department, The 198th Military Hospital, Chen Zhou 423000, PR China
Department of Pharmacy, Tangdu Hospital, The Fourth Military Medicine University, Xi’an 710038, Shaanxi, PR China
Department of Radiology, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, PR China
General Surgery Department, Tangdu Hospital, The Fourth Military Medical University, Xi’an 710038, PR China
b
c
d
e
f
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study, we firstly reported the antioxidant, antitumor and immunomodulatory effects of two
polysaccharides (GUMP-1-1 and GUMP-1-2) isolated from Grifola umbellata mycelia. Chemical analysis
indicated that two polysaccharide fractions contained different content of neutral sugar, uronic acid and
protein, as well as varying monosaccharide compositions and average molecular weight. We found that
they could significantly inhibit the growth of H22 implanted tumor and enhance the spleen index and
splenocyte proliferation of H22 tumor-bearing mice. In addition, GUMP-1-2 had the stronger free radicals
scavenging and ferrous ion chelating abilities than GUMP-1-1 in vitro. These results indicated that antitu-
mor activity of two purified polysaccharides might be achieved by improving immune response and the
different chemical composition and average molecular weight could affect their antitumor, antioxidant
and immunomodulatory activities.
Received 27 January 2013
Received in revised form 26 February 2013
Accepted 3 March 2013
Available online 13 March 2013
Keywords:
Grifola umbellate
Mycelia
Polysaccharide
Antioxidant
Anti-hepatoma
Immunomodulatory
© 2013 Elsevier Ltd. All rights reserved.
1
. Introduction
Grifola umbellata, a fungus that belongs to Polyporaceae, Basid-
medicinal materials and the harvest rate is exceedingly rapid than
that of rebirth speed by species themselves. Because of a lack of
effective protection and strong consumer demand, wild G. umbel-
lata resources have been seriously destroyed in China. So, there is
an urgent demand for a feasible alternative to obtain the bio-active
polysaccharides. Fortunately, this situation has been found and
much effort has been made done, mainly focusing on domestic cul-
tivation. Since the solid-state culture could not provide high yields
of bio-active polysaccharides, the submerged culture becomes the
only approach to produce mycelia and polysaccharides on a larger
scale. Recent studies have shown that the main ingredients of
the polysaccharides extracted from G. umbellata sclerotia and sub-
merged culture mycelia are the same (Xu, Li, Li, Liang, & Jin, 2004).
In spite of numerous studies on G. umbellate polysaccharide, there
are no available reports regarding its anti-hepatoma potential in
vivo. In this study, we aimed to investigate whether G. umbel-
late mycelia polysaccharides could inhibit mouse hepatoma H22
engrafted tumor in a rat model, and then to preliminarily investi-
gate its antioxidant and immunopotentiating activities.
iomycetes, is widely distributed in China, Japan, Europe and North
America. Its fruiting bodies growing above-ground is a delicious
edible mushroom and its sclerotium growing under-ground has
been used as a traditional Chinese medicine for the treatments
of inhibited urination, beriberi, edema and other diseases for cen-
turies (Zhou, Shao, & Chen, 2009). Polysaccharides isolated from G.
umbellata have been reported to the main bioactive components
to enhance human immunity and to treat certain cancers (Han,
1
988; Haranaka et al., 1985; Miyazaki & Oikawa, 1973; Miyazaki,
Oikawa, Yamada, & Yadomae, 1978; Miyazaki et al., 1979; Ueno,
Okamoto, Yamauchi, & Kato, 1982; You, Hau, Chen, & Huang, 1994).
Particularly, G. umbellata polysaccharide is effective in inhibiting
tumor growth. Moreover, many clinical studies show the therapeu-
tic alliance of G. umbellata polysaccharides and chemical medicines
can enhance the therapeutic efficacy and reduce the side effects
of the chemical counterparts (Lv, Liu, & Ning, 2005; Zhang et al.,
2
007). These reports greatly stimulate the consumption of this wild
2. Materials and methods
2.1. Materials and chemicals
∗ Corresponding author. Tel.: +86 29 84777432; fax: +86 29 84777432.
G. umbellata mycelia were from Hubei Chaihong Pharmaceuti-
cal Co. Ltd., China. DEAE-52 cellulose and Sephacryl S-400 were
E-mail address: wqfmmu@yahoo.com.cn (Q. Wang).
These authors contributed equally to this work.
1
0
144-8617/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carbpol.2013.03.002

Y. Bi et al. / Carbohydrate Polymers 95 (2013) 740–745
741
obtained from GE Healthcare Bio-Sciences Co., Piscataway, NJ, USA.
T-series Dextran (MW standards), ethylene diamine tetra-acetic
acid (EDTA), ascorbic acid, concanavalin A (Con A) and lipopolysac-
charide (LPS) were purchased from Sigma, Chemical Co., Saint Louis,
MO, USA. RPMI-1640 medium and fetal calf serum (FCS) were pur-
chased from Gibco, Grand Island, NY, USA. Aqueous solutions were
prepared with ultra-pure water from a Milli-Q water purification
system (Millipore, Bedford, MA, USA). All the other chemicals used
were of analytical grade.
Total neutral sugar content was determined by the reaction
with phenol in the presence of sulfuric acid at 486 nm (Dubois,
Gilles, Hamilton, Rebers, & Smith, 1956) using glucose as standard.
Protein was determined by photometric assay (Bradford, 1976)
using bovine serum albumin as the standard. Uronic acid was
determined by measuring the absorbance at 525 nm using the m-
hydroxybiphenyl photometric procedure, with d-glucuronic acid
as the standard (Blumenkrantz & Asboe-Hansen, 1973).
2
2
.4. Assay for antioxidant activity
2.2. Extraction, isolation and purification of polysaccharide
.4.1. Hydroxyl radical scavenging activity
The hydroxyl radical scavenging capacity was examined using
Briefly, the dried mycelia (200 g) were preliminarily treated
the method described previously (Chen, Ju, Li, & Yu, 2012). For the
control, sample was substituted by ascorbic acid. All values were
determined in three replicates. The percentage of hydroxyl radi-
cal scavenging capacity was calculated according to the following
equation:
with 1000 mL of 80% ethanol for 24 h to remove the interfering
components, such as monosaccharide, disaccharide, oligosaccha-
◦
ride and polyphenol in the samples at 80 C. After filtration, the
◦
residue was extracted twice with hot distilled water at 90 C for
4
h. The decoction was left to cool at room temperature, filtered
and then freeze-dried to obtain crude polysaccharides. The crude
polysaccharide was treated by Sevag reagent (Staub, 1965) to
remove protein and extensively dialyzed (cut-off Mw 3500 Da).
The retentate portion was then concentrated and centrifuged to
remove insoluble material. Finally the supernatant was lyophilized
to obtain crude polysaccharide, name as GUMP.
A532(blank) − A532(sample)
Scavenging effect (%) =
× 100
A5
32(blank)
where A532(blank) was the absorbance of the control (deionized
water, instead of sample) and A532(sample) was the absorbance of
the test sample mixed with reaction solution.
The GUMP was purified sequentially by chromatography of
DEAE-52 cellulose and Sephacryl S-400 column chromatography.
Briefly, 5 mL of GUMP solution (10 mg/mL) was applied to a col-
umn of DEAE-52 cellulose, and the column was stepwise eluted
with 0, 0.2, 0.4 and 0.6 M sodium chloride solutions at a flow rate
of 60 mL/h. Eluate was collected automatically (8 mL/tube), and
the carbohydrates were determined by the phenol–sulfuric acid
method. As a result, two fractions of polysaccharides (GUMP-1 and
GUMP-2) were obtained, concentrated, dialyzed against distilled
water and further purified through a column of Sephacryl S-400
2
.4.2. Superoxide anion scavenging activity
The superoxide anion scavenging capacity was examined using
the method described previously (Chen et al., 2012). For the control,
sample was substituted by ascorbic acid. All values were deter-
mined in three replicates. The percentage of superoxide anion
scavenging capacity was calculated according to the following
equation:
A560(blank) − A560(sample)
Scavenging effect (%) =
× 100
(
2× 60 cm), affording GUMP-1-1 and GUMP-1-2, respectively. The
A560(blank)
two purified fractions were collected, concentrated, dialyzed and
lyophilized for further study, respectively.
where A562(blank) was the absorbance of the control (deionized
water, instead of sample) and A562(sample) was the absorbance of
the test sample mixed with reaction solution.
2.3. Molecular weight and monosaccharide composition analysis
2
.4.3. Metal chelating assay
The molecular weight of the polysaccharide was determined
The chelating activity of sample on Fe2+ was measured as
by high performance gel permeation chromatography (HPGPC).
The sample solution was applied to Waters High Performance
Liquid Chromatography (HPLC) equipped with a TSK-GEL G3000
SWXL column (7.8 mm × 300 mm), eluted with 0.1 mol/L Na SO
reported (Wang, Yang, & Wei, 2012) by measuring the formation of
ferrous iron–ferrozine complex. For the control, sample was sub-
stituted with EDTA. The chelating activity was calculated as:
2
4
solution at a flow rate of 0.6 mL/min and detected by a Waters
A562(blank) − A562(sample)
Chelating ability (%) =
× 100
2
414 Refractive Index Detector. The columns were calibrated with
A
562(blank)
Dextran T-series standard of known molecular weight (200,000,
where A562(blank) was the absorbance of the control (deionized
water, instead of sample) and A562(sample) was the absorbance of
the test sample mixed with reaction solution.
7
1
0,000, 40,000, 10,000, 5000 Da). The molecular weight of GUMP-
-1 and GUMP-1-2 was estimated by reference to the calibration
curve made above.
◦
The polysaccharide was hydrolyzed by 2 M TFA at 120 C for 3 h
into monosaccharides under airtight condition, and the monosac-
charides were conventionally converted into the alditol acetates
as described (Xin et al., 2012) and were analyzed by gas chro-
matography (GC, Agilent 6890, USA) equipped with a HP-5 column
2.5. In vivo antitumor test
BALB/c mice (half male and half female, 18–22 g) were pur-
chased from Animal Experimental Center of the Fourth Military
Medical University. The animals were maintained on a 12-h-
(
30 m × 0.25 mm × 0.25 ␮m) and flame-ionization detector (FID).
◦
The operation was performed using the following conditions:
1
0
as the carrier gas at 1 mL/min; injection temperature was 250 C;
detector temperature was 300 C. Monosaccharides identification
dark/12-h-light cycle at 25 ± 1 C, humidity of 50 ± 10% and allowed
◦
◦
◦
◦
◦
60 C for 2 min, then to 200 C at 6 C/min, then to 215 C at
free access to standard laboratory pellet diet and water during
the experiments. Animal experiments were conducted under prin-
ciples in good laboratory animal care, and approved by ethical
committee for Laboratory Animals Care and Use of the Fourth Mil-
itary Medical University.
◦
◦
.2 C/min, then to 240 C at 6 C/min for 30 min. Nitrogen was used
◦
◦
was done by comparison with reference monosaccharides. The rel-
ative molar proportions were calculated by the area normalization
method.
Under sterile condition, 0.2 mL of ascites H22 tumor cell sus-
7
pension (1 × 10 cells/mL) was subcutaneously inoculated into the

7
42
Y. Bi et al. / Carbohydrate Polymers 95 (2013) 740–745
right armpit of BALB/c mice at day 0. Twenty-four hours after inoc-
ulation, mice were divided randomly into six groups (20 mice in
each group): model control group, positive control group and four
treatment groups. The mice inoculated with H22 cells were orally
administrated with two polysaccharides (100 and 200 mg/kg body
weight) or 5-FU (20 mg/kg) once daily for 10 days. The mice in
model control group received an equal volume of normal saline
(
0.2 mL/10 g body weight). Tumor volume was measured with
2
a digital caliper and calculated using the formula 0.52 × a × b ,
where a and b are the largest and smallest diameters as described
previously (Cai et al., 2008). Twenty-four hours after last tested
administration, all the tumor-bearing mice were sacrificed by cervi-
cal dislocation on day 11, and then tumor and spleen were collected
and weight. The rest 10 mice in each group were kept to evaluate
survival time. The inhibitory rate against the growth of tumor, the
spleen index and life prolongation rate were calculated according
to the following formula: the inhibitory rate (%) = [(the average vol-
ume of the model control group − the average volume of treated
groups)/the average volume of the model control group] × 100;
spleen index (mg/10 g) = (weight of spleen/body weight); life pro-
longation rate (%) = [(the survival time of the treated groups − the
survival time of the model control group)/the survival time of the
model control group] × 100. Body weights were measured at the
start and at the last day of treatment.
2.6. Splenocyte proliferation assay
Spleens collected from sacrificed mice under aseptic condition
were chopped into small pieces and passed through a fine steel
mesh to obtain a homogeneous cell suspension. Recovered spleen
cells were resuspended in lysis buffer (0.15 M NH Cl, pH 7.4) for
4
5
min to remove erythrocytes. Then spleen cells were harvested
and resuspended in RPMI 1640 complete medium. Cell numbers
and viability (over 95%) were assessed microscopically using try-
pan blue dye exclusion technique. The splenic cells (100 ␮l) were
Fig. 1. (A) Elution curve of crude polysaccharide extracted from Grifola umbellata
mycelia on a DEAE-52 cellulose anion-exchange column. (B) Purification of polysac-
charide fractions GUMP-1 and GUMP-2 on a Sephacryl S-400 column.
6
seeded in 96-well plates (1 × 10 cells/well) together with Con A
(
3
2.5 ␮g/ml) or LPS (5.0 ␮g/ml). After the cells were cultured at
7 C in a humidified atmosphere containing 5% CO2 for 48 h, 20 ␮l
◦
two fractions of GUMP-1-1 and GUMP-1-2 as detected by the
phenol–sulfuric acid assay (Fig. 1B). The physicochemical proper-
ties of two polysaccharides were summarized in Table 1. GUMP-1-1
had a negative response to the Bradford method, and no absorption
was detected by the UV spectrum at 280 and 260 nm, indicating the
absence of protein and nucleic acid. However GUMP-1-2 contained
a large amount of uronic acid (12.5%) and protein (13.6%), indicat-
ing GUMP-1-2 is an acid protein–polysaccharide. Accordingly, the
total sugar content evaluated in GUMP-1-1 and GUMP-1-2 were
MTT (5 mg/ml) were added to each well and incubated for another
h. After the incubation, the cell suspensions were centrifuged
at 800 rpm for 10 min and the supernatants were removed, then
50 ␮l of dimethyl sulfoxide (DMSO) working solution was added.
4
1
The absorbance was measured in an automatic ELISA plate reader
at 570 nm with a 630 nm reference after 30 min. The stimulation
index (SI) was calculated based on the following formula: SI = the
absorbance value for mitogen-cultures divided by the absorbance
value for non-stimulated cultures.
9
2.3% and 75.1%, respectively.
High-performance gel permeation chromatography (HPGPC)
2
.7. Statistical analysis
was used to determine the purity and molecular weight of the
polysaccharide. Each polysaccharide showed only one single sym-
metrical peak on HPGPC, suggesting that they were homogenous
All results were presented as mean ± SD. Data were analyzed by
one-way ANOVA using SPSS and Dunnett’s test. p values less than
0
3
3
.05 were considered significant.
Table 1
Components of monosaccharide and properties of two polysaccharide fractions from
Grifola umbellata mycelia.
. Results and discussion
Fractions
GUMP-1-1
GUMP-1-2
.1. Isolation, purification and composition of two
Total sugar (%)
Uronic acid (%)
Protein (%)
92.3
–
–
75.1
12.5
13.6
0.5
polysaccharides fractions
The crude polysaccharide (GUMP) was isolated from the hot
water extract of G. umbellata mycelia, followed by ethanol precip-
itation, deproteinization and lyophilization. After being fractioned
on DEAE-Sepharose CL-6B column, GUMP-1 (6.34%) and GUMP-2
5
Average molecular weights (10 Da)
2.5
Sugar components (mol%)
Glucose
Mannose
Galactose
Fructose
17.4
1.4
1.2
0.4
–
11.4
1.2
1.1
0.2
2.1
(
5.45%) were obtained from the distilled water and NaCl eluent,
respectively (Fig. 1A). GUMP-1 and GUMP-2 were further puri-
fied by Sephadex G-100 gel permeation chromatography, affording
Glucuronic acid

Y. Bi et al. / Carbohydrate Polymers 95 (2013) 740–745
743
polysaccharides. The average molecular weights of GUMP-1-1 and
GUMP-1-2 were calculated by the calibration curve with standard
5
T-series Dextrans to be 2.5 and 0.5 × 10 Da, respectively.
According to GC analysis, GUMP-1-1 was composed of glu-
cose, mannose, galactose and fructose in the molar ratios of
1
7.4:1.4:1.2:0.4. GUMP-1-2 contained glucose, mannose, galactose,
fructose and GlcA in the molar ratios of 11.4:1.2:1.1:0.2:2.1. These
data revealed glucose was the main neutral monosaccharide incor-
porated in two biopolymers, followed by mannose, galactose and
fructose.
3
3
.2. The antioxidant activity of two polysaccharides fractions
.2.1. Scavenging ability on hydroxyl radicals
Hydroxyl radical is the most reactive oxygen radical known
in chemistry, which can severely damage almost any biological
molecule in living cells and induce severe damage to the adja-
cent biomolecules. It is considered to be involved in aging, and the
development of cancer and several other diseases (Valentão et al.,
2
003). Thus, removal of hydrogen peroxide is very important for
protection of cells from oxidative damage. The scavenging abilities
of GUMP-1-1 and GUMP-1-2 on hydroxyl free radical are shown in
Fig. 2A. The scavenging rates of the GUMP-1-2 and ascorbic acid
against hydroxyl radicals were demonstrated in a dose-dependent
manner (Fig. 3A). Moreover, the neutral polysaccharide GUMP-1-1
exhibited weaker scavenging effect than the acidic polysaccharide
GUMP-1-2 at every concentration point. All these confirmed that
the uronic acid content and average molecular weight have signif-
icant effect on scavenging hydroxyl radical (Chen et al., 2012; Hu,
Liu, Chen, Wu, & Wang, 2010; Yu et al., 2003).
3.2.2. Scavenging ability on superoxide radicals
Superoxide anion, which is a relatively weak oxidant formed
in viable cells during several biochemical reactions, can be magni-
fied by its effect on cell damage because it can generate dangerous
species of free radicals and oxidizing agent (Chen et al., 2012). As
shown in Fig. 2B, a significant increase of the scavenging activity
on superoxide radicals was observed at the concentration range
(
0–8 mg/mL) of GUMP-1-2. Especially at the dose of 8 mg/mL, the
scavenging potency of GUMP-1-2 on superoxide radicals was close
to that of ascorbic acid. However GUMP-1-1 containing no uronic
acid showed a little or no scavenging activity on superoxide radicals
compared with GUMP-1-2 and ascorbic acid. This demonstrated
that the uronic acid content and average molecular weight affected
their antioxidant activities, which was in accordance with the fact
that higher uronic acid content and small molecular weight showed
greater scavenging effect on superoxide radical (Chen et al., 2012;
Hu et al., 2010; Yu et al., 2003).
3
.2.3. Chelating effect on ferrous ions
Fig. 2. The antioxidant activity of two polysaccharides fractions from Grifola umbel-
lata mycelia in vitro. (A) Hydroxyl radicals scavenging abilities of GUMP-1-1 and
GUMP-1-2; (B) superoxide radicals scavenging abilities of GUMP-1-1 and GUMP-1-
Transition metals, such as ferrous iron (Fe2+), can facilitate the
generation of ROS. Thus the chelating capacity of polysaccharides
2; (C) chelating abilities of GUMP-1-1 and GUMP-1-2 to ferrous ions. The values are
2
+
against Fe can be an important approach for retarding metal-
catalyzed oxidation (Hu et al., 2010). As shown in Fig. 2C, the ferrous
ion chelating ability climbed following a rise in polysaccharide con-
centration, with a peak level being attained for GUMP-1-2 (96.5%)
at 8 mg/mL. Conversely, a poor ferrous ion chelating activity was
found for GUMP-1-1 at all tested concentrations. It is worth point-
ing out that the compounds with structures containing two or more
presented as mean ± S.D. (n = 3).
3.3. Effect of two polysaccharides fractions on tumor growth and
survival time in H22-bearing mice
To investigate the anti-tumor activity of two polysaccharides
fractions in vivo, the mouse hepatoma H22 transplanted model was
created by subcutaneous injection of H22 cells into BALB/c mice.
The inhibitory effects of GUMP-1-1 and GUMP-1-2 are shown in
Fig. 3A. GUMP-1-1 and GUMP-1-2 obviously reduced the tumor
volume of H22 tumor-bearing mice, exhibiting the inhibition rate
of 45.2% and 63.2%, respectively at the dose of 200 mg/kg, which
of the following functional groups: OH, SH, COOH, PO H , C,
3
2
O, NR2,
ration can show metal chelating activity (Yuan, Bone, & Carrington,
005). Similarly, a lack of carboxyl group may lead to a lower metal
S
and
O
in a favorable structure–function configu-
2
ion chelating capacity of GUMP-1-1 at every concentration point
compared to acidic polysaccharide GUMP-1-2.

7
44
Y. Bi et al. / Carbohydrate Polymers 95 (2013) 740–745
Fig. 3. Effect of two polysaccharides fractions from Grifola umbellata mycelia on
the tumor volume and the survival time of H22-bearing mice. (A) Inhibition rate
of tumor growth; (B) life prolonged rate. The values are presented as mean ± S.D.
(
n = 10). Significant differences compared to negative control group are designated
*
*
***
as p < 0.01 and p < 0.001.
Fig. 4. Effect of two polysaccharides fractions from Grifola umbellata mycelia on
spleen index and concanavalin A (Con A)- and lipopolysaccharide (LPS)-stimulated
splenocyte proliferation of H22-bearing mice. (A) Spleen index; (B) splenocyte pro-
liferation index (SI). The values are presented as mean ± S.D. (n = 10). Significant
indicated that GUMP-1-1 and GUMP-1-2 had significant antitumor
activity in vivo against H22 tumor. More importantly, GUMP-1-1
and GUMP-1-2 significantly prolonged the survival period of H22-
bearing mice, showing the life prolonged ratios of 56.3% and 85.8%,
respectively at the dose of 200 mg/kg (Fig. 3B). As expected, 5-FU
significantly prolonged the survival period of H22-bearing mice and
suppressed tumor generation of the H22-tumor bearing mice. In
addition, no significant loss of body weight, anorexia, listlessness,
abdominal distention, liver function, renal function or other appar-
ent adverse effects were observed among the animals, with the
exception of 5-FU treated group (data not shown).
*
differences compared to negative control group are designated as p < 0.05 and
*
*
p < 0.01.
by GUMP-1-1 or GUMP-1-2 at the doses of 100 and 200 mg/kg
compared to the model control (p < 0.05 and p < 0.01) (Fig. 4B).
4. Conclusions
In the paper, two main polysaccharide fractions (GUMP-1-
1
and GUMP-1-2) were purified from G. umbellata mycelia by
fractionation on DEAE-52 cellulose and Sephacryl S-400 column
chromatography. Chemical analysis indicated that two polysaccha-
ride fractions contained different contents of neutral sugar, uronic
acid and protein, as well as varying monosaccharide compositions
and average molecular weight. These properties provided basis
for the differences of antioxidant, antitumor and immunopoten-
tiating activities of these polysaccharide fractions. Several in vitro
assays, such as hydroxyl radical scavenging assay, superoxide rad-
ical scavenging assay, and chelating effects on ferrous ion, were
applied to evaluate the antioxidant potential of the two polysac-
charides. Among two fractions, GUMP-1-2 had the stronger free
radicals scavenging and ferrous ion chelating abilities than GUMP-
1-1, implying some factors including the content of uronic acid,
protein content and molecular weight may influence their antiox-
idant activities. Furthermore, GUMP-1-1 and GUMP-1-2 could
significantly inhibit the growth of H22 tumor in BALB/c mice
and extended the survival time of mice bearing H22 tumor.
3
.4. Effect of two polysaccharides fractions on spleen index and
splenocyte proliferation in H22-bearing mice
It
is
reported
that
the
anticancer
activities
of
polysaccharide–protein complexes could be associated with
their immunostimulating properties (Ooi & Liu, 2000). To inves-
tigate whether two polysaccharides fractions enhance humoral
immunity or cell-mediated immunity, we measured the effect
of GUMP-1-1 and GUMP-1-2 on spleen index and mitogen-
stimulated splenocyte proliferation in tumor-bearing mice. As
shown in Fig. 4A, an increase in relative spleen weight of GUMP-1-1
or GUMP-1-2-treated groups was observed, compared with control
group (p < 0.05 and p < 0.01). While spleen index of 5-FU-treated
group decreased significantly (p < 0.05). Furthermore Con A- and
LPS-induced splenocyte proliferation was significantly enhanced

Y. Bi et al. / Carbohydrate Polymers 95 (2013) 740–745
745
GUMP-1-1 and GUMP-1-2 could also remarkably increase the
spleen weights and splenocyte proliferation of H22 tumor bear-
ing mice, which indicated that they could improve the immune
response. Interestingly, a high inhibition ratio of H22 tumor growth
was observed in GUMP-1-2-treated group at very dose point com-
pared to that of GUMP-1-2-treated group. This different antitumor
potency probably depended on their monosaccharide composi-
tion, protein content, molecular mass and chain conformation.
The above results suggested that the antitumor activity of these
two polysaccharides might be achieved by improving immune
response. However, the antitumor mechanisms of GUMP-1-1 and
GUMP-1-2 remain unclear. Further studies are needed to elucidate
the structure-function relationships and mechanisms responsible
for its antitumor activities.
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Acknowledgement
This research was supported by the National Natural Science
Foundation of China (No. 81072050).
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