Antitumor and immunomodulatory activities of a water-soluble polysaccharide from Chaenomeles speciosa

Article abstract of DOI:10.1016/j.carbpol.2015.06.046

Abstract In this study, a water-soluble polysaccharide (CSP) was successfully purified from Chaenomeles speciosa by DEAE-Sepharose and Sephadex G-100 column chromatography. CSP had a weight-average molecular weight of about 6.3 × 10⁴ Da and was composed of glucose (Glc), galactose (Gal), rhamnose (Rha) and arabinose (Ara) with a relative molar ratio of 4.6:1.3:0.8:0.5. CSP could not only inhibit the growth of S180 tumor transplanted in mice, but also increase the relative spleen index and body weight of tumor bearing mice. Moreover, concanavalin A (ConA) and lipopolysaccharide (LPS) induced splenocyte proliferation and peritoneal macrophage phagocytosis were also enhanced after CSP administration. Furthermore, CSP treatment could improve delayed type hypersensitivity (DTH) and promote the secretion of IL-2, TNF-α and IFN-γ in serum. The overall findings suggest that the antitumor effect of CSP is might be associated with its potent immunostimulatory activity.

Full text of DOI:10.1016/j.carbpol.2015.06.046

Carbohydrate Polymers 132 (2015) 323–329
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Antitumor and immunomodulatory activities of a water-soluble
polysaccharide from Chaenomeles speciosa
a,b,∗
b
b
Xianfei Xie
, Guolin Zou , Chenghai Li
a
Department of Pharmacology, Basic Medical School, Wuhan University, Wuhan 430072, PR China
College of Life Sciences, State Key Laboratory of Virology, Wuhan University, Wuhan 430072, PR China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 April 2015
Received in revised form 8 June 2015
Accepted 9 June 2015
Available online 20 June 2015
In this study, a water-soluble polysaccharide (CSP) was successfully purified from Chaenomeles speciosa
by DEAE-Sepharose and Sephadex G-100 column chromatography. CSP had a weight-average molecular
4
weight of about 6.3 × 10 Da and was composed of glucose (Glc), galactose (Gal), rhamnose (Rha) and
arabinose (Ara) with a relative molar ratio of 4.6:1.3:0.8:0.5. CSP could not only inhibit the growth of S180
tumor transplanted in mice, but also increase the relative spleen index and body weight of tumor bearing
mice. Moreover, concanavalin A (ConA) and lipopolysaccharide (LPS) induced splenocyte proliferation
and peritoneal macrophage phagocytosis were also enhanced after CSP administration. Furthermore, CSP
treatment could improve delayed type hypersensitivity (DTH) and promote the secretion of IL-2, TNF-␣
and IFN-␥ in serum. The overall findings suggest that the antitumor effect of CSP is might be associated
with its potent immunostimulatory activity.
Keywords:
Chaenomeles speciosa
Polysaccharides
Antitumor
Immunomodulation
©
2015 Elsevier Ltd. All rights reserved.
1
. Introduction
Most polysaccharides derived from higher plants are relatively
nontoxic and do not cause significant side effects compared with
synthetic compounds, and hence they are ideal candidates for
modern medicine (Wang, Chen, Jia, Tang, & Ma, 2012; Zhang, Li,
Xiong, Jiang, & Lai, 2013). Therefore, discovery and evaluation of
polysaccharides with antitumor and immunostimulating prop-
erties have emerged as one of the important research fields in
chemistry and biology (Fan, Ding, Ai, & Deng, 2012; Li et al., 2012).
Polysaccharides from C. speciosa have been found to be involved
in antioxidant and antiinflammatory activities (Li & Chen, 2011;
Liu, Wang, Lu, Ma, & Zhang, 2011a). However the antitumor and
immunoregulatory properties of water-soluble polysaccharides
from C. speciosa have not previously been investigated, in this
regard the objectives of this study are to elucidate the isolation and
characterization of the biological polysaccharide from C. speciosa
and evaluate its antitumor and immunomodulatory activities on
the immune response in tumor-bearing mice by using both in vitro
and in vivo assay.
Chaenomeles speciosa (Sweet) Nakai (Rosaceae) is a well-known
food in China, and its dried fruit is commonly used in traditional
medicine (Jiangsu New Medical College, 1977). Modern studies
have shown that C. speciosa has a variety of biological and pharma-
cological activities such as immunomodulatory, antiinflammatory,
antitumor, antimicrobial and antioxidant effects (Dai, Wei, Shen, &
Zheng, 2003; Xie, Cai, Zhu, & Zou, 2007; Zhang et al., 2010). Evidence
has suggest that carbohydrates, amino acids, proteins, tannins and
other organic acids are the main ingredients of C. speciosa fruits
(Chen & Wei, 2003; Chen, Wu, & Dai, 2000; Li & He, 2005).
Cancer is one of the major causes of human death worldwide.
The great majority of chemical compounds, which have been
identified as cytotoxic to cancer cells, are also toxic to normal cells
(Borchers, Stern, Hackman, Keen, & Gershwin, 1999). Recently,
research interest has focused on polysaccharides from various
plants and herbs with immune-stimulating properties, which
may be useful complementary or alternative medicine in helping
humans to reduce the risk of cancer as well as infectious disease.
2. Materials and methods
2.1. Materials and reagents
∗ Corresponding author at: Department of Pharmacology, Basic Medical School,
Wuhan University, Wuhan 430072, PR China. Tel.: +86 27 68758665;
fax: +86 27 68759222.
Dried fruits of C. speciosa were collected from Changyang in
Hubei Province (China). A voucher specimen was deposited in the
herbarium of College of Life Sciences, Wuhan University, China.
E-mail address: Xiexianfeiut@gmail.com (X. Xie).
http://dx.doi.org/10.1016/j.carbpol.2015.06.046
0
144-8617/© 2015 Elsevier Ltd. All rights reserved.

3
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X. Xie et al. / Carbohydrate Polymers 132 (2015) 323–329
Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetra zolium bromide (MTT), trifluoroacetic acid (TFA),
(Liu et al., 2011a; Liu et al., 2011b). CSP (10 mg) were hydrolyzed
with 2 M TFA at 100 C for 2 h, and converted to their alditol acetates
◦
concanavalin
A
(ConA),
lipopolysaccharide
(LPS),
2,4-
as previously described (Honda, Suzuki, Kakehi, Honda, & Takai,
1981). The resulting alditol-acetates was analyzed on an Agilent
6280 instrument fitted with FID and equipped with a HP-5 fused
silica capillary (0.25 mm × 30 m × 0.25 ␮m). The temperature of the
dinitrofluorobenzene (DNFB), standard sugars and T-series
dextran were purchased from Sigma Chemical Co. (St. Louis, MO,
USA). RPMI 1640 medium and bovine serum albumin (BSA) were
purchased from Gibco (Grand Island, NY, USA). Cyclophosphamide
◦
◦
column was kept at 150 C for 10 min and then increased to 250 C
◦
(
CTX) was purchased from Jiangsu Hengrui Co. (Lianyungang,
at the rate of 5 C/min subsequently hold on 5 min. The rate of N2
China). DEAE-Sepharose and Sephadex G-100 were purchased
from Amersham Pharmacia Co. (Sweden). All other chemical
reagents used were analytical grade.
carrier gas was 1 ml/min.
2.3.4. FT-IR spectral analysis
The FT-IR spectrum of CSP was recorded with a Nicolet 6700 FT-
IR Spectrometer (Thermo Co., USA). The dried sample was ground
with potassium bromide powder and pressed into pellet for spec-
2
.2. Extraction, isolation and purification of the polysaccharide
−
1
Dried powdered fruits (200 g) were extracted by hot water
trometric measurement in the frequency range of 4000–400 cm
.
under stirring for 5 h in a boiling-water bath (at the ratio of 1:20,
w/v) and repeated twice. The aqueous extract was filtered, and the
polysaccharides were precipitated from the filtrates by 95% ethanol
2.4. Cytotoxicity on Sarcoma 180 cells in vitro
(
1:4, v/v), then keeping it overnight to ensure the precipitates is
The cytotoxicity of CSP on Sarcoma 180 (S180) cells was evalu-
ated by the colorimetric MTT method (Mosmann, 1983). S180 cells
(obtained from China Center for Type Culture Collection (CCTCC),
Wuhan University) were prepared from peritoneal cavity of the
tumor inoculated mice under aseptic conditions. Then the cells
◦
complete. After standing out overnight at 4 C, it was centrifuged for
3
0 min at 4500 × g. The precipitated materials were collected and
dissolved in water to sufficiently mix with Sevag reagent for remov-
ing the free protein and combined protein (Staub, 1965) in this
precipitate. The sample was then dialysed against distilled water
and precipitated by four volumes of 95% ethanol. The precipitate
was collected by centrifugation, washed successively with ethanol,
acetone and petroleum ether and lyophilized to obtain the crude
polysaccharide (21 g).
4
were plated in a 96-well plates at a density of 1 × 10 cells/ml
◦
in RPMI-1640 medium and after 24 h incubation at 37 C they
were exposed to various concentrations (50, 100 and 200 ␮g/ml)
of polysaccharide for 24, 48 and 72 h. After then, 10 ␮l of MTT
(0.5 mg/ml) was added to each well and the cells were cultured
for another 4 h. The supernatant was removed by centrifuging
and 100 ␮l DMSO was added into each well for the dissolution of
formazan crystals. The absorbance was measured at 570 nm on a
micro-plate Reader (Bio-rad, USA). The antitumor activity of the
tested samples was expressed as an inhibition ratio (percent) calcu-
The crude polysaccharide (8 g) was redissolved in distilled water
(
100 ml) and applied to a DEAE-Sepharose Fast-Flow chromatogra-
phy column (2.6 × 40 cm), and eluted stepwise with distilled water
and a gradient of 0.1–0.5 M NaCl at a flow rate of 1 ml/min. Guided
by the phenol–sulfuric acid method, the water eluting fraction
with high content of sugar was collected, dialyzed, lyophilized, and
purified by Sephadex G-100 (2.6 × 100 cm) gel-permeation chro-
matography eluted with 0.1 M NaCl at a flow rate of 0.5 ml/min.
One purified polysaccharide fraction obtained was named as CSP.
lated as: [1 − (A5 of treated cells/A570 of untreated cells) × 100%].
70
All tests were run in triplicate.
2.5. Animal groups and in vivo antitumor test
2
.3. Analysis of purified polysaccharide
Male Kunming mice (20 ± 2) g was provided by the Laboratory
Animal Center, Wuhan University, China. The animals were housed
◦
2.3.1. Chemical properties analysis
under normal laboratory conditions (21 ± 2 C, 12/12 h light/dark
Total carbohydrate content of polysaccharide was determined
cycle). The animals were given a standard laboratory diet and water
ad libitum. All animal (used for this experiment) handling pro-
cedures were performed in strict compliance with the PR China
legislation, with the guidelines established by the Institute for
Experimental Animals of Wuhan University, and were approved
by the University committee for animal experiments.
by phenol–sulfuric acid colorimetric method using glucose as
the standard (Dubois, Gills, Hamilton, Rebers, & Smith, 1956).
The protein contents of the purified polysaccharides were mea-
sured according to Bradford’s method, using BSA as the standard
(Bradford, 1976), and the uronic acid content assessed using the
Blumenkrantz and Asboe-Hansen method (Blumenkrantz & Asboe-
Hansen, 1973).
Under sterile condition, 0.2 ml of S180 cell suspension was
subcutaneously inoculated into Kunming mice (1 × 10 cells/ml).
6
The mice inoculated were divided into five groups (ten mice per
group): 50, 100, 200 mg/kg CSP treatment groups, positive controls
(20 mg/kg CTX) and negative controls (physiological saline). Daily
orally (p. o.) drug administration was begun 24 h after the inoc-
ulation and performed once daily for 10 consecutive days. After
stopping administration, mice were weighed and sacrificed by cer-
vical dislocation. Tumors and spleens were excised and weighted,
respectively. Spleen index was expressed in the spleen weight rel-
ative to body weight. The tumor inhibitory rate was calculated by
the following formula: the inhibition ratio (%) = [(A − B)/A] × 100,
where A is the average tumor weight of the negative control group,
and B is that of the treatment group (Sun et al., 2009).
2
.3.2. Homogeneity and average molecular weight
The homogeneity and molecular weight of CSP were determined
by high-performance gel-permeation chromatography (HPGPC)
on LC-10A liquid chromatography instrument (Shimadzu,
a
Japan). TSK-Gel G4000 column (7.8 mm ID × 30 cm) was main-
◦
tained at 35 C and the mobile phase was 0.1 M Na SO (flow
2
4
rate = 0.5 ml/min), and detected by a RID-10A detector. The sample
2 mg) was dissolved in the mobile phase and was filtered through
(
a 0.45 ␮m filter. The average molecular weight was estimated by
reference to the calibration curve made from a Dextran T-series
standard of known molecular weight (T-10, T-40, T-70, T-500 and
T-2000).
2.6. Spleen lymphocyte proliferation assay
2.3.3. Monosaccharide composition analysis
The identification and quantification of the monosaccharides of
Splenocytes taken from mice of all the groups were suspended
polysaccharide were achieved by gas chromatography (GC) analysis
in RPMI-1640 medium. Splenocytes was plated in a 96-well culture

X. Xie et al. / Carbohydrate Polymers 132 (2015) 323–329
325
Fig. 1. Elution profiles of crude polysaccharide from the dried fruits of C. speciosa on anion-exchange chromatography column of DEAE-Sepharose (A) and gel filtration
chromatography column of Sephadex G-100 (B).
plate with or without ConA (5 ␮g/ml) or LPS (10 ␮g/ml) at 2 × 106
cells/ml in 200 ␮l complete medium. The plates were incubated
box, fixed with acetone–methanol (1/1, v/v) solution, and then
stained by 4% (v/v) Giemsa-phosphoric acid dye. The number of
macrophage ingesting CRBC out of a total of at least 100 cells was
calculated by direct visual counting using a light microscope. The
phagocytic rate (PR) was calculated using the following formula:
PR (%) = number of macrophage ingesting CRBC/total number of
macrophages × 100.
◦
at 37 C in a humidified atmosphere with 5% CO . After 44 h, MTT
2
(
20 ␮l, 2 mg/ml) was added to each well and the plates were incu-
bated for another 4 h. After aspirating the supernatant from the
wells, 100 ␮l of DMSO was added to dissolve formazan crystals. The
absorbance of spleen lymphocytes cells in each well were measured
at 570 nm by an ELISA reader (Bio-Rad, USA) (Mosmann, 1983).
2.8. Delayed-type hypersensitivity reaction to DNFB (DTH)
2.7. Macrophage phagocytosis assay
After 5 days of oral administration as Section 2.5, mice were
sensitized to DNFB by smearing 50 ␮l 1% DNFB in acetone–castor
oil (v/v = 1:1) on the shaved abdominal of which was unhaired by
Phagocytosis of peritoneal macrophages was measured accord-
ing to the reported method with some modification (Lin, Feng,
Pan, Zhang, & Xiao, 1995). The first 10 days of oral administra-
tion were the same for Section 2.5, on the last day, all the mice
received 0.5 ml of 10% (v/v) chicken red blood cells (CRBC) by
intraperitoneally injection, and the mice were euthanized by cervi-
cal dislocation 1 h later. 2 ml saline was injected into the abdominal
cavity and 1 ml fluid was then collected to make a smear for each
3
1
× 3 cm. Five days later, the DTH reaction was elicited by smearing
0 ␮l 1% DNFB on both sides of the left ear, while the right unpainted
earlap was used as control. Twenty-four hours later, the mice were
sacrificed by cervical dislocation; the DTH response to DNFB was
evaluated by measuring the weight difference of the right and left
ear with an analytical balance (Brodmerkel et al., 2005).
◦
mouse. The smears were incubated at 37 C for 30 min in a wet

3
26
X. Xie et al. / Carbohydrate Polymers 132 (2015) 323–329
Table 1
Inhibitory effect of CSP on S180 cells in vitro.
Group
Concentration
␮g/ml)
Inhibition rate (%)
(
24 h
48 h
72 h
CSP
CSP
CSP
50
100
200
1.86 ± 0.19
2.73 ± 0.24
3.13 ± 0.37
2.20 ± 0.19
3.85 ± 0.37
3.44 ± 0.35
2.07 ± 0.22
4.42 ± 0.39
4.16 ± 0.40
Each value is presented as mean ± S.D. (n = 3).
at 1369 cm 1 was assigned to C H bending vibration. The absorp-
−
−
1
tions in the range of 1000–1200 cm , attributed to the stretching
vibrations of C C and C H, were observed.
O
O
3.2. In vivo and in vitro antitumor activities
Fig. 2. FT-IR spectrum of CSP.
The growth inhibition effect of CSP was tested on S180 cells by
MTT assay in vitro. As shown in Table 1, the inhibition ratio on S180
cells of all CSP groups (50, 100 and 200 ␮g/ml) at different time
was lower than 5%, suggesting that CSP had no direct cytotoxicity
against S180 cells.
The antitumor effects of CSP were examined in the S180 tumor-
bearing mice in vivo at the dose of 50, 100 and 200 mg/kg. As
shown in Table 2, after daily p. o. administration of CSP for 10
days, we found that CSP significantly inhibited tumor growth in a
dose-dependent manner, with the inhibitory rates of 28.2%, 37.0%,
and 44.9%, at the concentrations of 50, 100 and 200 mg/kg respec-
tively and tumor inhibitory rates treated by 20 mg/kg CTX was
2
.9. Serum IL-2, TNF-˛ and IFN-ꢀ determination
The serum collected from a different group was detected
for interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-␣) and
interferon-gamma (IFN-␥) level using a commercial ELISA kit
according to the instructions of kits (Shanghai Qianchen Biotech
Co., China). The absorbance was measured at 450 nm in an ELISA
reader (Bio-Rad, USA) (Wang et al., 2007).
2.10. Statistical analysis
5
9.0%. At the same time, the spleen index was increased obviously
Experimental results were expressed as mean ± standard devia-
in CSP administration (P < 0.05), whereas CTX treatment slightly
decreased the index compared to the control group. The relative
spleen weight was an important index for nonspecific immunity,
which suggested that CSP may inhibit tumor growth by strong
stimulatory effects to the immunological function of S180 tumor-
bearing mice.
tion (S.D.). Statistical analysis was performed using Student’s t-test.
The significant difference was set at p < 0.05.
3
. Results and discussion
3.1. Purification and characteristics of the polysaccharide
3.3. Effects of the polysaccharide on splenocyte proliferation
The crude polysaccharide was gained from the dried fruit of
C. speciosa, and the yield was about 5.28% of the dry material. As
shown in Fig. 1A, it was separated by using anion-exchange chro-
matography on DEAE-Sepharose column, which was successively
eluted with distilled water and a gradient of 0.1–0.5 M NaCl. Two
fractions of polysaccharides were obtained, designated as CCSP-
The effect of CSP on mitogen-stimulated splenocyte prolifera-
tion in S180 tumor-bearing mice was presented in Fig. 3. ConA-
and LPS-induced splenocyte proliferation in S180 tumor-bearing
mice was significantly enhanced by CSP at the doses of 50, 100 and
200 mg/kg in a dose-dependent manner (P < 0.05 or P < 0.01), but
splenocyte proliferations in the CTX-treated group were slightly
suppressed than those of the control group.
Lymphocyte proliferation plays a crucial role in the cellular
immune responses. Lymphocytes proliferation induced by ConA or
LPS may be used as a method to evaluate T or B lymphocyte activity
(Han et al., 1998). T and B lymphocytes are two important classes of
immunologically active cells. The former is mainly responsible for
cellular immunity, and the latter is the only cell capable of produc-
ing antibodies (Mosmann & Coffman, 1989). The results indicated
1
and CCSP-2. The CCSP-1 was applied to a Sephadex G-100 gel
filtration column for further purification. The column was eluted
with 0.1 M NaCl, and the resulting elute was collected. As shown
in Fig. 1B, the fraction generated one single elution peak, afford-
ing CSP. CSP appeared as a light-yellow powder and contains 95.4%
of total sugar as determined by the phenol-sulfuric acid method. It
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. As determined by m-
hydroxydiphenyl colorimetric method, the polysaccharide did not
contain uronic acid.
Table 2
A single and symmetrically sharp peak observed on HPGPC indi-
cated that CSP was a homogeneous polysaccharide. According to
the calibration curve with standard dextrans, the average molecu-
Effect of CSP on tumor growth and spleen index in S180 tumor-bearing mice.
Group
Dose (mg/kg) Tumor weight (g) Inhibition Spleen index (mg/g)
rate (%)
4
lar weight of it was estimated to be about 6.3 × 10 Da. Analysis by
Control
CSP
CSP
CSP
CTX
2.27 ± 0.23
1.63 ± 0.16
1.43 ± 0.15
1.25 ± 0.12
0.93 ± 0.09
6.76 ± 0.58
8.13 ± 0.67
9.45 ± 0.82
11.12 ± 1.06
5.95 ± 0.49
GC indicated that it was composed of glucose (Glc), galactose (Gal),
rhamnose (Rha) and arabinose (Ara) with a relative molar ratio of
a
a
b
b
a
a
b
50
100
200
20
28.2
37.0
44.9
59.0
4
.6:1.3:0.8:0.5.
The FT-IR spectrum of CSP was illustrated in Fig. 2. The broad
−
1
characteristic intense peak around 3407 cm was due to the O
H
Each value is presented as mean ± S.D. (n = 10).
stretching vibration of the polysaccharide. The band in the region
a
P < 0.05 compared to control group.
P < 0.01 compared to control group.
−
1
b
of 2925 cm was due to C H stretching vibration and the band

X. Xie et al. / Carbohydrate Polymers 132 (2015) 323–329
327
Fig. 3. Effect of CSP on splenocyte proliferation in mice. Each value is presented
as mean ± S.D. (n = 10). *P < 0.05 compared to control group. **P < 0.01 compared to
control group.
Fig. 5. Effects of CSP on the swelling rate of earlap in mice. Each value is presented
as mean ± S.D. (n = 10). *P < 0.05 compared to control group. **P < 0.01 compared to
control group.
that CSP could significantly promote the Con A- and LPS-stimulated
splenocyte proliferation in tumor-bearing mice, suggesting that
immunomodulation might be its anti-tumor mechanism.
3
.5. Effect of the polysaccharide on DTH response
Fig. 5 showed the effects of CSP on the DNFB-induced DTH
of mice. A significant increase in the swelling rate of earlap was
observed in CSP-treated group, especially at the dose of 200 mg/kg,
compared with control group (P < 0.01). However, DTH reaction was
obviously suppressed by CTX administration, with respect to the
control group (P < 0.05).
DTH, is a cell-mediated pathologic response involved in T cell
activation and cytokine production (Dai, Zhang, Zhang, & Wang,
2009). The character of DTH reaction is the large aggregation of non-
specific inflammatory cells, in which the macrophage is a major
participant. It is a type IV hypersensitivity reaction that develops
when an antigen activates sensitized TDTH cells (Umesh, Hawna,
Paramjit, Dinesh, & Suresh, 2004). Treatment of CSP enhanced DTH
reaction, which was reflected from the increased earlap swelling
compared to control group suggesting heightening infiltration of
macrophages to the inflammatory site. This result may also be sup-
porting a possible role of CSP in assisting the cell-mediated immune
response.
3
.4. Effects of the polysaccharide on macrophage phagocytosis
The effect of CSP treatment on macrophage phagocytosis in S180
tumor-bearing mice was shown in Fig. 4, a considerable enhance-
ment of the proliferation of peritoneal macrophages was observed
in CSP treated groups (50, 100 and 200 mg/kg) when compared to
the control group (P < 0.05 or P < 0.01). In the meantime, CTX appar-
ently inhibited macrophage phagocytosis because of its side effect
to the immune system (P < 0.01).
As the most important professional phagocyte, macrophage
is essential for maintaining homeostasis regardless of varying
external conditions and considered to be one of the important com-
ponents of the host defense against tumor growth (Gamal-Eldeen,
Amer, Helmy, Talaat, & Ragab, 2007; Katsiari, Liossis, & Sfikakis,
2
010). Phagocytic capacity is one of the most important indica-
tors of the body’s non-specific immunity (Schepetkin & Quinn,
006). It protects the host by phagocytosis, presents antigens to
2
lymphocytes and releases numerous cell factors that regulate the
activity of other cells (Jiao et al., 2009). The results of the study
indicated that CSP could significantly improve the phagocytic activ-
ity of macrophages in S180 tumor-bearing mice, which means the
improving of immune state of the host.
3
.6. Effect of the polysaccharide on IL-2, TNF-˛ and IFN-ꢀ
production
Cytokines are peptides or proteins with low molecular weights,
which affect cell functions and condition their interactions. They
are produced by cells that possess regulatory properties and are
found very close to or in direct contact with target cells. By binding
to specific cell surface receptors, they affect cell proliferation, differ-
entiation and functions. Cytokines exert an effect on hematopoiesis
and immune processes including acute phase reaction and antitu-
mor defense. They regulate both cellular and humoral responses
(Terlikowski, 2002).
Several polysaccharides derived from plants have been reported
to induce production of cytokines including IL-2, TNF-␣ and IFN-␥
Schepetkin & Quinn, 2006). In this study, the effects of CSP on the
(
secretion of serum IL-2, TNF-␣ and IFN-␥ in S180 tumor-bearing
mice were examined by using ELISA kits. As shown in Table 3, CSP
significantly augmented the secretion of serum IL-2, TNF-␣ and IFN-
␥
as compared with the model control, especially at the doses of
1
00 mg/kg/day and 200 mg/kg/day (P < 0.01). On the contrary, the
levels of serum IL-2, TNF-␣ and IFN-␥ in the CTX-treated group
were much lower than those of the model control (P < 0.01). IL-2 is
essential for the growth, proliferation, and differentiation of T cells,
and is produced by T cells normally during an immune response
(Malek, 2008). TNF-␣ is a cytokine with tumor necrosis activity that
Fig. 4. Effect of CSP on macrophage phagocytosis in mice. Each value is presented
as mean ± S.D. (n = 10). *P < 0.05 compared to control group. **P < 0.01 compared to
control group.

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X. Xie et al. / Carbohydrate Polymers 132 (2015) 323–329
Table 3
Effect of CSP on serum IL-2, TNF-␣ and IFN-␥ levels in mice.
Group
Dose (mg/kg)
IL-2 (pg/ml)
TNF-␣ (pg/ml)
IFN-␥ (pg/ml)
Control
CSP
CSP
CSP
CTX
48.72 ± 4.13
57.36 ± 6.24
68.21 ± 6.77
78.32 ± 8.76
26.18 ± 2.64
238.44 ± 20.42
288.16 ± 27.13
345.79 ± 32.62
410.23 ± 39.75
110.32 ± 10.56
172.71 ± 14.52
233.28 ± 21.75
287.42 ± 30.14
345.59 ± 34.82
a
b
b
b
b
b
b
b
b
b
b
50
100
200
20
b
92.76 ± 9.89
Each value is presented as mean ± S.D. (n = 10).
a
P < 0.05 compared to control group.
P < 0.01 compared to control group.
b
is secreted mainly by macrophages and has been recognized as an
important host regulatory molecule (Vilcek & Lee, 1991). IFN-␥ is
mainly secreted by NK cells and T cells as part of innate immunity
and antigen-specific immunity (Boehm, Klamp, Groot, & Howard,
Dai, M., Wei, W., Shen, Y. X., & Zheng, Y. Q. (2003). Glucosides of Chaenomeles
speciosa remit rat adjuvant arthritis by inhibiting synoviocyte activities. Acta
Pharmacologica Sinica, 24, 1161–1166.
Dai, Z. Y., Zhang, H., Zhang, Y. P., & Wang, H. H. (2009). Chemical properties and
immunostimulatory activity of a water-soluble polysaccharide from the clam of
Hyriopsis cumingii Lea. Carbohydrate Polymers, 77, 365–369.
1
997). It demonstrated that CSP could enhance the immune func-
Dubois, M., Gills, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric
method for determination of sugars and related substances. Analytical Chemistry,
tion by promoting cytokine levels in S180 tumor-bearing mice.
2
8, 350–356.
Fan, L. P., Ding, S. D., Ai, L. Z., & Deng, K. Q. (2012). Antitumor and immunomodulatory
activity of water-soluble polysaccharide from Inonotus obliquus. Carbohydrate
Polymers, 90, 870–874.
4
. Conclusion
Gamal-Eldeen, A. M., Amer, H., Helmy, W. A., Talaat, R. M., & Ragab, H. (2007). Chemi-
cally -modified polysaccharide extract derived from Leucaena leucocephala alters
Raw 264.7 murine macrophage functions. International Immunopharmacology, 7,
In the present study, a polysaccharide (CSP) from C. speciosa
was obtained by hot water extraction and then purified by DEAE-
Sepharose anion-exchange and Sephadex G-100 gel-permeation
chromatography. The CSP was a homogeneous polysaccharide with
871–878.
Honda, S., Suzuki, S., Kakehi, K., Honda, A.,
&
Takai, T. (1981). Analysis of
the monosaccharide compositions of total non-dialyzable urinary glyco-
conjugates by the dithioacetal method. Journal of Chromatography, 226,
341–350.
4
a molecular weight of 6.3 × 10 Da, as determined by HPGPC.
GC analysis showed that CSP was composed of glucose, galac-
tose, rhamnose and arabinose with a relative molar ratio of
Han, S. B., Kim, Y. H., Lee, C. W., Park, S. M., Lee, H. Y., Ahn, K. S., et al. (1998). Char-
acteristic immunostimulation by angelan isolated from Angelica gigas Nakai.
Immunopharmacology, 40, 39–48.
4
.6:1.3:0.8:0.5. Furthermore, CSP had indirect anti-tumor activ-
ity achieved by improving immune response in vivo, and it might
act as a potential natural anti-tumor agent with immunomodula-
tory activity. It has been demonstrated that many factors such as
monosaccharide composition affected the activities of polysaccha-
rides (Bao, Liu, Fang, & Li, 2001), and polysaccharides composed of
glucan have been proved to stimulate the immune system (Kuang,
Xia, Yang, Wang, & Wang, 2011). The immunostimulatoryactivityof
CSP might be due to its high contents of glucose. Further researches
about elucidating the detailed mechanism of its antitumor actions
are in process.
Jiangsu New Medical College. (1977). Dictionary of Chinese Materia Medical Science
and Technology. Shanghai: Press of Shanghai.
Jiao, L. L., Li, X., Li, T. B., Jiang, P., Zhang, L. X., Wu, M. J., et al. (2009). Characterization
and anti-tumor activity of alkali-extracted polysaccharide from Enteromorpha
intestinalis. International Immunopharmacology, 9, 324–329.
Katsiari, C. G., Liossis, S. C., & Sfikakis, P. P. (2010). The pathophysiologic role of
monocytes and macrophages in systemic lupus erythematosus: A reappraisal.
Seminars in Arthritis and Rheumatism, 39, 491–503.
Kuang, H., Xia, Y., Yang, B., Wang, Q., & Wang, Y. (2011). Screening and comparison of
the immunosuppressive activities of polysaccharides from the stems of Ephedra
sinica Stapf. Carbohydrate Polymers, 83, 787–795.
Li, D., & He, L. (2005). Determination of olenolic acid and urolic acid in Fructus
chaenomelis by HPLC-evaporative light-scattering detection method. Chinese
Journal of Hospital Pharmacy, 25, 259–261.
Li, S. G., & Chen, Y. (2011). Effect of Chaenomeles speciosa polysaccharide on adjuvant
arthritis in mice and its mechanisms. Chinese Journal of Experimental Traditional
Medical Formulae, 12, 159–162.
Acknowledgments
Li, X. L., Wang, Z. H., Zhao, Y. X., Luo, S. J., Zhang, D. W., Xiao, S. X., et al. (2012).
Isolation and antitumor activities of acidic polysaccharide from Gynostemma
pentaphyllum Makino. Carbohydrate Polymers, 89, 942–947.
The authors wish to thank Mr. Zhang Tao for growing and col-
lecting plant material. This work was supported by the National
Natural Science Foundation of China, through grant No. 81001617.
Lin, P. Y., Feng, Z. M., Pan, J. Q., Zhang, D.,
artesunate on immune function in mice. Zhong guo Yao Li Xue Bao, 16,
41–444.
Liu, J., Wang, W., Lu, K., Ma, L.,
& Xiao, L. Y. (1995). Effects of
4
&
Zhang, T. X. (2011). Extraction and
References
antioxidation activity of polysaccharides from Chaenomeles speciosa (Sweet.)
Nakai. Journal of Henan University of Technology (Natural Science Edition), 1,
4
8–52.
Bao, X., Liu, C., Fang, J., & Li, X. (2001). Structural and immunological studies of a
major polysaccharide from spores of Ganoderma lucidum (Fr.) Karst. Carbohy-
drate Polymers, 332, 67–74.
Blumenkrantz, N., & Asboe-Hansen, G. (1973). New method for quantitative deter-
mination of uronic acids. Analytical Biochemistry, 54, 484–489.
Boehm, U., Klamp, T., Groot, M., & Howard, J. C. (1997). Cellular responses to
interferon-gamma. Annual Review of Immunology, 15, 749–795.
Borchers, A. T., Stern, J. S., Hackman, R. M., Keen, C. L., & Gershwin, M. E. (1999).
Mushrooms, tumors, and immunity. Proceeding of Society of Experimental Biology
and Medicine, 221, 281–293.
Liu, J., Zhang, C., Wang, Y., Yu, H., Liu, H., Wang, L., et al. (2011). Structural elucidation
of a heteroglycan from the fruiting bodies of Agaricus blazei Murill. International
Journal of Biological Macromolecules, 49, 716–720.
Malek, T. R. (2008). The biology of interleukin-2. Annual Review of Immunology, 26,
453–479.
Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival:
Application to proliferation and cytotoxicity assays. Journal of Immunological
Methods, 16, 55–63.
Mosmann, T., & Coffman, R. (1989). TH1 and TH2 cells: Different patterns of lym-
phokine secretion lead to different functional properties. Annual Review of
Immunology, 7, 145–173.
Schepetkin, I. A., & Quinn, M. T. (2006). Botanical polysaccharides: Macrophage
immunomodulation and therapeutic potential. International Immunopharmacol-
ogy, 6, 317–333.
Staub, A. M. (1965). Removal of protein-Sevag method. Methods in Carbohydrate
Chemistry, 5, 5–6.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of micro-
gram quantities of protein utilizing the principle of protein binding. Analytical
Biochemistry, 72, 248–254.
Brodmerkel, C. M., Huber, R., Covington, M., Diamond, S., Hall, L., Collins, R., et al.
(
2005). Discovery and pharmacological characterization of a novel rodent-active
CCR2 antagonist, INCB3344. The Journal of Immunology, 175, 5370–5378.
Chen, Q., & Wei, W. (2003). Effects and mechanisms of glucosides of Chaenomeles
speciosa on collagen-induced arthritis in rats. International Immunopharmacol-
ogy, 3, 593–608.
Chen, R. L., Wu, T. J., & Dai, Y. J. (2000). Studies on the chemical constituents of four
species of Chaenolemes. West China Journal of Pharmaceutical Sciences, 15, 38–39.
Sun, Y. X., Li, Y. J., Li, M. Q., Tong, H. B., Yang, X. D., & Liu, J. C. (2009). Optimiza-
tion of extraction technology of the Anemone raddeana polysaccharides (ARP)
by orthogonal test design and evaluation of its anti-tumor activity. Carbohydrate
Polymers, 75, 575–579.

X. Xie et al. / Carbohydrate Polymers 132 (2015) 323–329
329
Terlikowski, S. J. (2002). Local immunotherapy with rhTNF-␣ mutein induces strong
antitumor activity without overt toxicity - A review. Toxicology, 174, 143–152.
Umesh, T., Hawna, R., Paramjit, S., Dinesh, K. S., & Suresh, P. V. (2004). Immunomod-
ulatory effects of aqueous extract of Tridax procumbens in experimental animals.
Journal of Ethnopharmacology, 92, 113–119.
Wang, R., Chen, P., Jia, F., Tang, J., & Ma, F. (2012). Optimization of polysaccharides
from Panax japonicus C.A, Meyer by RSM and its anti-oxidant activity. Interna-
tional Journal of Biological Macromolecules, 50, 331–336.
Xie, X. F., Cai, X. Q., Zhu, S. Y., & Zou, G. L. (2007). Chemical composition and antimicro-
bial activity of essential oils of Chaenomeles speciosa from China. Food Chemistry,
100, 1312–1315.
Zhang, D., Li, S., Xiong, Q., Jiang, C., & Lai, X. (2013). Extraction, characterization and
biological activities of polysaccharides from Amomum villosum. Carbohydrate
Polymers, 95, 114–122.
Zhang, L., Cheng, Y. X., Liu, A. L., Wang, H. D., Wang, Y. L., & Du, G. H. (2010).
Antioxidant, anti-inflammatory and anti-influenza properties of components
from Chaenomeles speciosa. Molecules, 15, 8507–8517.
Vilcek, J., & Lee, T. H. (1991). Tumor necrosis factor, New insights into the molecu-
lar mechanisms of its multiple actions. The Journal of Biological Chemistry, 266,
7
313–7316.
Wang, G., Zhao, J., Liu, J. W., Huang, Y. P., Zhong, J. J., & Tang, W. (2007). Enhancement
of IL-2 and IFN-gamma expression and NK cells activity involved in the anti-
tumor effect of ganoderic acid Me in vivo. International Immunopharmacology, 7,
8
64–870.