Purification, characterization and immunomodulating activity of a polysaccharide from flowers of Abelmoschus esculentus

Article abstract of DOI:10.1016/j.carbpol.2014.02.079

A water-soluble polysaccharide (OFPS11) was obtained from okra (Abelmoschus esculentus) flowers using aqueous extraction and purification with DEAE-52 cellulose and Sephacryl S-500 column. Its preliminary characterization and immunomodulating activity were investigated. Results showed that OFPS11 is mainly composed of galactose and rhamnose in a molar ratio of 2.23:1 with molecular mass of 1700 kDa. RAW264.7 cells pretreated with OFPS11 significantly inhibited the proliferation of HepG-2 cells. Additionally, OFPS11 enhanced the phagocytic ability and induced the elevation of NO production, TNF-α and IL-1β secretion of RAW264.7 cells. Furthermore, OFPS11 promoted both the expression of iNOS protein and of iNOS and TNF-α mRNA. OFPS11 can strongly increase NF-κB levels in nucleuses, which is an important transcription factor that can modulate expressions of iNOS, NO and TNF-α. These outcomes support that OFPS11 exerts its antitumor activity by probably stimulating macrophage activities through nuclear NF-κB pathway.

Full text of DOI:10.1016/j.carbpol.2014.02.079

Carbohydrate Polymers 106 (2014) 335–342
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Purification, characterization and immunomodulating activity of a
polysaccharide from flowers of Abelmoschus esculentus
Wei Zhenga,1, Ting Zhaob,1, Weiwei Fengc, Wei Wangc, Ye Zouc, Daheng Zhengc,
Mohammed Takaseb, Qian Lia, Huiyu Wua, Liuqing Yangb,∗, Xiangyang Wud,∗∗
a School of Pharmacy, Jiangsu University, Xuefu Rd. 301, Zhenjiang 212013, Jiangsu, China
b School of Chemistry and Chemical Engineering, Jiangsu University, Xuefu Rd. 301, Zhenjiang 212013, China
c School of Food and Biological Engineering, Jiangsu University, Xuefu Rd. 301, Zhenjiang 212013, China
d School of the Environment, Jiangsu University, Xuefu Rd. 301, Zhenjiang 212013, Jiangsu, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A water-soluble polysaccharide (OFPS11) was obtained from okra (Abelmoschus esculentus) flowers using
aqueous extraction and purification with DEAE-52 cellulose and SephacrylTM S-500 column. Its prelimi-
nary characterization and immunomodulating activity were investigated. Results showed that OFPS11 is
mainly composed of galactose and rhamnose in a molar ratio of 2.23:1 with molecular mass of 1700 kDa.
RAW264.7 cells pretreated with OFPS11 significantly inhibited the proliferation of HepG-2 cells. Addi-
tionally, OFPS11 enhanced the phagocytic ability and induced the elevation of NO production, TNF-␣
and IL-1␤ secretion of RAW264.7 cells. Furthermore, OFPS11 promoted both the expression of iNOS pro-
tein and of iNOS and TNF-␣ mRNA. OFPS11 can strongly increase NF-␬B levels in nucleuses, which is an
important transcription factor that can modulate expressions of iNOS, NO and TNF-␣. These outcomes
support that OFPS11 exerts its antitumor activity by probably stimulating macrophage activities through
nuclear NF-␬B pathway.
Received 26 January 2014
Received in revised form 19 February 2014
Accepted 28 February 2014
Available online 11 March 2014
Keywords:
Okra flowers
Polysaccharide
Immunomodulating activity
Structural characterization
Macrophage activation
© 2014 Elsevier Ltd. All rights reserved.
1. Introduction
elderly people and cancer patients. Studies have demonstrated
that the potential immune-stimulating activity and antitumor
Many plant active ingredients, especially polysaccharides, have
gained much popularity on account of their broad spectrum of
biological and pharmacological activities, such as antitumor (Cai
et al., 2012b; Shen et al., 2012; Wu, Zhu et al., 2012; Zhao et al.,
2014), immune-modulatory (Yang, Hsieh, Lu & Lin, 2013; Zhao
et al., 2013), antiviral (Jiang et al., 2013; Oliveira et al., 2013), anti-
complementary (Di, Zhang & Chen, 2013; Hromádková, Paulsen,
Polovka, Kosˇt’álová & Ebringerová, 2013), antioxidant (Fan et al.,
2011; Mao et al., 2014; Xu, Xiao & Mao, 2013; Zhao et al., 2012), and
anti-inflammatory (Liao & Lin, 2013) activities. Among these, inter-
est is growing recently in the antitumor and immune-stimulating
activity of natural polysaccharides due to their relatively low tox-
icities and few side effects (Bai et al., 2012; Cai et al., 2012a).
Immune-stimulating is regarded as an important strategy which
is used to enhance the body’s defense mechanism, especially for
property are generally related to macrophage activation and mod-
ulation of complement system (Han et al., 2006; Kim et al., 2007;
Shang et al., 2003). Activation of macrophages can enhance their
antitumor activity through secretion of cytokines or by antigen
processing and presentation, thereby regulating the immune sys-
tem (Klimp, De Vries, Scherphof & Daemen, 2002). Thus, it may
be one of therapeutical interests to explore new polysaccharides
as immune-potentiator to stimulate the macrophage-mediated
defense mechanisms.
Okra (Abelmoschus esculentus L.), an indigenous crop in Africa
(Molfetta, Ceccarini, Macchia, Flamini & Cioni, 2013), is now widely
distributed in various parts of the world (Jarret, Wang & Levy,
2011). Published data indicated that okra fruit exhibited promising
immune-potentiating effects owing to its thick and slimy polysac-
charide (Sheu & Lai, 2012). Due to the high content of similar
mucilaginous, okra flowers are therefore expected to be good
sources of immune-stimulating polysaccharides. Although numer-
ous activities of polysaccharide from okra fruits have been reported
so far, studies have not been reported on the isolation, structural
characterization and immune-regulation of polysaccharide from
okra flowers. Therefore, the aim of the present study was to isolate
∗
Corresponding author. Tel.: +86 511 88791800; fax: +86 511 88791800.
Corresponding author. Tel.: +86 511 88791200; fax: +86 511 88791200.
E-mail addresses: yangliuqing@ujs.edu.cn (L. Yang), wuxy@ujs.edu.cn (X. Wu).
∗∗
1
These authors are contributed equally to this work and are co-first authors.
http://dx.doi.org/10.1016/j.carbpol.2014.02.079
0144-8617/© 2014 Elsevier Ltd. All rights reserved.

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W. Zheng et al. / Carbohydrate Polymers 106 (2014) 335–342
a SephacrylTM S-500 High Resolution (4000–2,000,000) gel col-
umn (1.6 cm × 50 cm) using 0.1 M NaCl as eluent at a flow rate
of 0.3 mL/min. One single fraction named OFPS11 was collected,
dialysis as above, and finally freeze-dried.
and preliminarily characterize a water-soluble polysaccharide from
okra flowers, evaluate its immune-stimulating effects on murine
RAW264.7 macrophage cells and explore whether immunopoten-
tiation is involved in its action. The study will be helpful to develop
novel functional foods and drugs.
2.4. Characterization of OFPS11
2. Materials and methods
2.4.1. Molecular weight determination
2.1. Materials and reagents
The homogeneity and molecular weight distribution of OFPS11
were determined by high-performance gel-permeation chro-
matography (HPGPC) on a LC-10ATvp instrument (Shimadzu,
Tokyo, Japan), equipped with a pre-column of TSKGUARD COLUMN
PWH (7.5 mm × 75 mm, Tosoh corporation, Tokyo, Japan) and a col-
umn of TSK-GEL G4000PW (7.5 mm × 300 mm, Tosoh corporation,
Tokyo, Japan) and eluted with 0.003 M CH3COONa solution at a flow
rate of 1.0 mL/min. The eluents were monitored using refractive
index detector (Shimadzu RID-10A, Shimadzu). Standard T-series
dextrans were used as standards for calibration (2000, 500, 70, 40
and 10 kDa).
Okra (Abelmoschus esculentus L.) flowers were collected from
Zhenjiang, Jiangsu Province in August 2012, and identified by Chen
Li (Chief Pharmacist, Food and Drug Administration Bureau of Zhen-
jiang). The moisture content of the fresh flowers was determined in
2 h after harvest. The flowers were packaged 500 g per polyethylene
bag and then stored at −20 ◦C until use. 3-(4,5-Dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethylsulfoxide
(DMSO) and lipopolysaccharide (LPS) were obtained from Sigma
Chemical Co. (St. Louis, MO, USA). Dulbecco’s modified Eagle’s min-
imum essential medium (DMEM) and fetal bovine serum (FBS) was
obtained from Gibco (Grand Island, NY, USA), respectively. Assay
kits for interleukin-1␤ (IL-1␤), tumor necrosis factor-alpha (TNF-
␣), nitric oxide (NO) test kits and BCA were obtained from Nanjing
Jiancheng Bioengineering Institute (Nanjing, Jiangsu Province,
China). Dextrans of different molecular weights (T-10, T-40, T-70, T-
500, and T-2000) were obtained from Pharmacia Co., Ltd. (Uppsala,
Sweden). DEAE-52 cellulose and SephacrylTM S-500 were obtained
from Whatman Co., Ltd. (Maidstone, Kent, UK). All other chemicals
and solvents used were of analytical reagent grade and obtained
from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
2.4.2. Neutral monosaccharide composition analysis
The monosaccharide compositions of OFPS11 were determined
using the method reported by (Yang et al., 2011) with slight mod-
ification. Briefly, the OFPS11 was hydrolyzed with 4.0 M H2SO4 at
110 ◦C for 10 h. After neutralization with BaCO3, the supernatants
were collected and lyophilized. The hydrolysates were then con-
verted into their corresponding alditol acetates and analyzed with
GC using a Shimadzu 2010 instrument, equipped with a HP-5MS
column (0.25 mm × 30 m × 0.25 ␮m) and a flame-ionization detec-
tor. The temperature was set as follows: the initial temperature of
column was 130 ◦C, maintained for 5 min and increased to 240 ◦C at
4 ◦C/min. The 240 ◦C was held for 5 min. Standard monosaccharides
were then converted to their acetylated derivative and analyzed
using the same procedure.
2.2. Cell culture
Human hepatocellular carcinoma HepG-2 and mouse
macrophage cell line RAW264.7 cells were obtained from Institute
of Cell Biology, Chinese Academy Sciences (Shanghai, China).
Cells were cultured at 37 ◦C in DMEM supplemented with 10%
FBS, penicillin (100 IU/mL), and streptomycin (100 ␮g/mL) in a
humidified atmosphere with 5% CO2 and 95% air.
2.4.3. Infrared spectra analysis
To detect functional groups of OFPS11, IR spectrum were
obtained with a NEXUS 670 FT-IR (Thermo Nicolet, USA) spec-
trophotometer. OFPS11 was grounded, mixed with KBr powder
(1:100), pressed into pellets and detected in the frequency range of
4000–400 cm−1
.
2.3. Extraction, isolation and purification of polysaccharides
500 g frozen sample was cut into small slices and homoge-
nized in double-distilled water (ddH2O) with a high-frequency
homogenizer. The mixture was transferred and extracted twice
with ddH2O for 3 h, and supernatants were collected by cen-
trifugation at 4500 rpm for 30 min. The combined filtrates were
concentrated and precipitated with 95% (v/v) alcohol until the con-
centration of the alcohol reached 80% and kept for 12 h at 4 ◦C, then
centrifuged again as above. The precipitated material was then dis-
solved and dialyzed through cellulose membrane (Sigma–Aldrich,
retaining > Mw 14,000) against flowing water and ddH2O for 48 h,
respectively. The aqueous solution was then collected from the
dialysis bag and freeze-dried to obtain the crude polysaccharide
(OFPS).
To remove the proteins, OFPS was re-dissolved and subjected
to the trichloroacetic acid (TCA) method (Lengsfeld, Titgemeyer,
Faller & Hensel, 2004). Then the de-proteined polysaccharides were
stepwise eluted with different concentrations of NaCl solutions
(0, 0.05, 0.1, 0.15, 0.2, 0.5, 1.0 M) by DEAE-52 (OH−) cellulose
column (1.6 cm × 50 cm). Forty test tubes (5 mL of each) were
monitored spectrophotometrically at 490 nm with phenol-sulfuric
acid method (Dubois, Gilles, Hamilton, Rebers & Smith, 1956)
and collected. One fraction (OFPS1), which was obtained in 0.1 M
NaCl, comprised the major components of the elution fraction and
was further fractionated with size-exclusion chromatography on
2.5. Macrophage immune-modulating activity
2.5.1. Cytotoxicity assay
The cytotoxic effects of OFPS11 on RAW264.7 cells were mea-
sured by MTT assay. In brief, adherent RAW264.7 cells in 96-well
plates (2 × 104 cells/well) were cultured with different concentra-
tions of OFPS11 (0–1000 ␮g/mL) prepared in DMEM (10% FBS). The
LPS (10 ␮g/mL) was used as a positive control (Wang et al., 2013).
The treated cells were incubated for 24 and 48 h to estimate the via-
bility. 1 mg/mL of MTT was prepared and 100 ␮L MTT was added
to each well and incubated for 4 h. After incubation, the observed
purple color of mazone crystals was dissolved with 100 ␮L DMSO
for 1 h. The absorbance was detected at 570 nm by microplate ELISA
reader (Spectra MAX 190, Molecular Devices Corporation, USA).
2.5.2. Macrophage-mediated cytotoxicity
Briefly, adherent RAW264.7 cells in 96-well plates
(2 × 104 cells/well) were cultured with different concentra-
tions of OFPS11 (0–1000 ␮g/mL) and LPS (10 ␮g/mL). After 48 h
incubation, the supernatants were collected by centrifugation at
1500 rpm for 15 min. Then the collected supernatants and treated
macrophages were analyzed, respectively. On the one hand, the
collected supernatants were added to another 96-well plates with
adherent HepG-2 cells (4 × 103/well). After culturing for 48 h, the

W. Zheng et al. / Carbohydrate Polymers 106 (2014) 335–342
337
Fig. 1. Flowchart scheme of macrophage-mediated cytotoxicity on HepG-2 cells.
supernatant-stimulated HepG-2 cells were then used for MTT
assay as described above. On the other hand, the OFPS11-treated
cells were rinsed twice with preheated DMEM (10% FBS) to remove
stimulants (as shown in Fig. 1). The stimulated RAW264.7 (effector
cells) were then co-incubated with 100 ␮L HpeG-2 cells suspension
(target cells, 2 × 104 cells/mL, 1:10 of the density of effector cells)
for another 48 h incubation. After incubation, an MTT assay was
used for measuring the inhibition of tumor cells.
membranes were treated with rabbit anti-iNOS monoclonal anti-
body (Santa Cruz Biotechnology, Inc., USA) and rabbit anti-␤-actin
(Santa Cruz Biotechnology, Inc., USA) overnight at 4 ◦C, respectively,
then the membranes followed by incubation with horseradish
peroxidase-conjugated anti-rabbit antibody (Santa Cruz) for 3 h at
room temperature. The blot was visualized using the ECL-detection
with chemiluminescence system. Analysis of NF-␬B using nuclear
extracts with ␤-actin as internal reference was also performed as
above. The lysis buffer contains 20 mM Tris (pH 7.5), 2 mM EDTA,
135 mM NaCl, 2 mM DTT, 2 mM sodium pyrophosphate, 25 mM ␤-
glycerophosphate, 10% glycerol, 1% Triton X-100, 1 mM Na3VO4,
10 mM NaF, 10 ␮g/mL leupeptin, 10 ␮g/mL aprotinin, and 1 mM
PMSF. The primary antibody was rabbit anti-NF-␬B p65 monoclonal
antibody (Santa Cruz Biotechnology, Inc., USA), and the secondary
antibody was horseradish peroxidase-conjugated anti-rabbit anti-
body.
2.5.3. Neutral red uptake by RAW264.7
Phagocytic activity was determined with neutral red uptake
assay. In brief, adherent RAW264.7 cells in 96-well plates
(2 × 104 cells/well) were treated with different concentrations of
OFPS11 (0–1000 ␮g/mL) and LPS (10 ␮g/mL) for 48 h, and then
the culture medium was abandoned. One hundred microliter of
0.075% (g/mL) neutral red solution was added to each well and cul-
tured for a further 4 h. After cultivation, the pretreated macrophage
was washed three times with phosphate buffered saline (PBS) and
100 ␮L of cell lysis buffer (acetic acid/ethanol; 1:1) was added and
incubated at 4 ◦C for 2 h. The absorbance was determined at 540 nm
using microplate ELISA reader.
2.5.6. RT-PCR
To evaluate the mRNA expression levels of inducible
nitric oxide synthase (iNOS) and TNF-␣, total RNA from
LPS and OFPS11-treated RAW264.7 cells were prepared by
adding RNAiso Plus (Takara, China), according to the man-
ufacturer’s instructions. RT-PCR was carried out by using
two-step RT-PCR System kit (Takara, China) with following
primer sequences: ꢀ5-CCCTTCCGAAGTTTCTGGCAGCAGC-3ꢀ (for-
ward) and ꢀ5-GGCTGTCAGAGCCTCGTGGCTTTGG-3ꢀ (reverse)
for mouse iNOS, ꢀ5-ATGAGCACAGAAAGCATGATC-3ꢀ (forward),
ꢀ5-TACAGGCTTGTCACTCGAATT-3ꢀ (reverse) for mouse TNF-␣,
2.5.4. Measurement of NO and cytokine
RAW264.7 cells were treated with OFPS11 in different con-
centrations and LPS (10 ␮g/mL) as above. After 48 h, the cultured
supernatants were collected for the nitrite assay with Nitric oxide
test kits (Kim et al., 2012; Zhang & Dai, 2011) and cytokine secre-
tion assay using an enzyme-linked immunosorbent assay (ELISA),
respectively. The concentration of nitrite was calculated using a
standard curve and cytokine concentration was measured follow-
ing the manufacturer’s instructions.
ꢀ5-TGGAATCCTGTGGCATCCATGAAAC-3ꢀ
(forward)
and
ꢀ5-
TAAAACGCAGCTCAGTAACAGTCCG-3ꢀ (reverse) for mouse ␤-actin.
The ␤-actin primer was used as an internal control. PCR was
performed with 1 cycle of 180 s at 94 ◦C, 25 cycles of 30 s at 94 ◦C,
40 s at 56 ◦C, 60 s at 72 ◦C and 1 cycle of 180 s at 72 ◦C in a 50 ␮L
reaction mixture, and the products were separated on a 1.5%
agarose gels and visualized by ethidium bromide staining. The gel
slabs were scanned and analyzed with image analysis software
(Kodak Digital Science, Kennesaw, GA, USA).
2.5.5. Western blot analysis
The effect of OFPS11 on iNOS protein expression in RAW264.7
cells was performed using whole-cell extracts. Cells treated with
OFPS11 (0–1000 ␮g/mL) and LPS (10 ␮g/mL) were rinsed twice
with ice-cold PBS, lysed using ice-cold extraction buffer (10 mM
HEPES, 150 mM NaCl, 1 mM EGTA, 1% CHAPS, and 1% Triton X-
100) containing 1% of protease inhibitor cocktail for 30 min at 4 ◦C.
Protein concentrations were determined using the BCA protein
assay kit. The protein extract was then boiled with isotonic loading
buffer for 10 min, and an equal amount of protein (20 ng) for each
sample was separated by a 12.5% SDS-PAGE and followed by electri-
cally transferring to polyvinylidene difluoride (PVDF) membranes.
After blocking with 5% skim milk in TBS-0.1% Tween 20 (TBST), the
2.6. Statistical analysis
All experiments were repeated at least three times. The statisti-
cal values were presented as mean standard deviation and datas
were analyzed by the Tukey method multiple comparison tests
to evaluate significant differences. A value of p < 0.05 denoted the

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W. Zheng et al. / Carbohydrate Polymers 106 (2014) 335–342
0.009
0.006
0.003
0.000
Volts
0
5
10
15
Minutes
Fig. 2. HPSEC chromatogram of OFPS11.
presence of a statistically significant difference. Statistical analyses
were carried out by SPSS version 16.0 (SPSS Inc, Chicago, USA).
3. Results and discussion
3.1. Isolation, purification and molecular weight of OFPS11
The moisture of the fresh okra flowers was (90.42 0.11) %, and
the yield of OFPS was calculated to be 15.4% by dry basis. After
TCA treatment, no absorption was detected by the UV spectrum
at 280 and 260 nm, which indicates the absence of protein and
nucleic acid. OFPS was then fractionated through DEAE-52 cellu-
lose and SephacrylTM S-500 to obtain a white fluffy polysaccharide
named OFPS11. The molecular weight of OFPS11 was determined
by HPGPC, which has been shown to be an effective method (López-
Barajas, López-Tamames & Buxaderas, 1998). As shown in Fig. 2, a
single and symmetrical peak indicates a high purity of the OPFS11
and the average molecular weight of OFPS11 was calculated as
1700 kDa according to the calibration curve.
3.2. Monosaccharide composition and infrared spectroscopy
Fig. 3. (A) Gas chromatograms of acetate-derivatized standard monosacharides and
monosaccharide compositions of OFPS11. (a) l-rhamnose (Rha), (b) l-arabinose
(Ara), (c) d-xylose (Xyl), (d) d-mannose (Man), (e) d-glucose (Glu) and (f) d-galactose
(Gal). (B) The FTIR spectra of OFPS11.
To further investigate the composition of OFPS11, GC was used.
As shown in Fig. 3A, the neutral sugars of OFPS11 was mainly com-
posed of galactose and rhamnose in a ratio of 2.23:1, and it also
contains some other sugars such as arabinose, mannose, and glu-
cose with little content. The infrared spectra (Fig. 3B) showed strong
and wide stretching peak around 3387.76 cm−1 for O H stretching
vibrations, a weak absorption peak at 2937.80 cm−1 was induced
by C H stretching vibration. The intense peak which appeared in
1611.67 cm−1 was the C O asymmetric stretching of COO, and
Tao, 2007). However, OFPS11 was not able to inhibit the prolifer-
ation of HepG-2 cells at the tested concentrations. Thus, it might
indicate that the potential antitumor activity of OFPS11 showed
in an indirect way, for example, by boosting the immune sys-
tem. The cytotoxicity effect of OFPS11 on RAW264.7 cells was
also determined. As seen in Fig. 4A, after treatment with OFPS11
(0–1000 ␮g/mL) and LPS (10 ␮g/mL) for 24 and 48 h, there were
disorderly changes in the OD values. The data demonstrated that
OFPS11 showed no significant effects on RAW264.7 cells up to
1000 ␮g/mL. Therefore, due to the hardly any cytotoxicity of
OFPS11 on RAW264.7 cells, these macrophages were selected as
test cells in the following study.
the band at 1421 cm−1 was attributed to bending vibration of C
H
or O H (Cao, Yuan, Sun & Sun, 2011). Particular polysaccharide has
specific bands in the 1000–1200 cm−1 region, and this region is
dominated by ring vibrations overlapped with stretching vibrations
of (C OH) side groups and the (C
O C) glycosidic band vibration.
Absorptions at 1146.99, 1095.39, 1073.56, and 1044.07 cm−1 indi-
cated a pyranose form of sugar (Wu, Mao et al., 2012; Zhang, Liu,
Park, Xia & Kim, 2012).
3.3. Cytotoxicity assay
3.4. Proliferation inhibition with OFPS11-M-CM and
macrophage-mediated cytotoxicity activity with OFPS11
Many kinds of polysaccharides (Jin, 2012; Zhao et al., 2012), as
natural biological macromolecules, have been proved to exhibit
significant antitumor activities with little toxicity. The cytotox-
icity of OFPS11 on HepG-2 cells was examined with MTT using
5-fluorouracil (5-FU) as the positive control (Shi, Nie, Chen, Liu &
Our study above indicated that OFPS11 exhibited an indi-
rect cyctotoxic activity against HepG-2 tumor cells. To investigate
whether macrophage-conditioned (M-conditioned) media induced
by different concentrations of OFPS11 could affect the proliferation

W. Zheng et al. / Carbohydrate Polymers 106 (2014) 335–342
339
Fig. 4. (A) Effects of OFPS11 on the proliferation of RAW264.7 macrophages. RAW264.7 cells (2 × 104 cells/well) were incubated with different concentrations of OFPS11
(0–1000 ␮g/mL) and LPS (10 ␮g/mL) for 24 h or 48 h. Effect of OFPS11-induced M-conditioned media (B) and OFPS11-activated macrophage (D) on the proliferation of HepG-2
cells. RAW 264.7 Cells (2 × 104 cells/well) were incubated with OFPS11 (0–1000 ␮g/mL) and LPS (10 ␮g/mL) for 48 h. Antitumor activity was determined as described in 2.5.2
at an initial effector/target ratio of 10:1. Each value is presented as mean SD of 5 separate experiments. P values are shown as ap < 0.05, aap < 0.01 compared with normal
control group. A value of p < 0.05 demoted the presence of a statistically significant difference according to Tukey method. (C) Effect of OFPS11 on phagocytosis of RAW264.7
macrophages by neutral red uptake assay. RAW264.7 cells (2 × 104 cells/well) were incubated with OFPS11 (0–1000 ␮g/mL) and LPS (10 ␮g/mL) for 48 h. The absorbance at
540 nm was determined.
of HepG-2 cells, an MTT assay was performed. As seen in Fig. 4B,
M-conditioned media induced by OFPS11 (62.5–1000 ␮g/mL)
inhibited the proliferation of HepG-2 cells in a distinct dose-
dependent manner. Moreover, the inhibition of all concentrations
was significant compared with normal control group.
at other concentrations was significant compared with the normal
control group. From the data, it is suggested that OFPS11 can play
action on phagocytic function of macrophages in some degree.
3.6. NO and cytokine levels
To assess the cytotoxic activity of OFPS11-activated
macrophages, the HepG-2 cells were co-incubated with OFPS11
and LPS-treated RAW264.7 respectively. From Fig. 4D, the anti-
tumor activity was observed in the OFPS11-induced macrophage
groups, and the higher concentrations (250–1000 ␮g/mL) of
OFPS11 significantly enhanced cytotoxic activity of RAW264.7
cells against HepG-2 cells compared to normal control group.
These results indicated that OFPS11 exhibited a great immuno-
logical competence by stimulating macrophage responses, and
the immune-modulating activity was one of the important
mechanisms in the antitumor activity of OFPS11.
As a significant part of host defense system, macrophages can
produce various mediators and cytokines, which plays potential
roles in defense against cancer cells (Bontà & Ben-Efraim, 1993).
NO, an intracellular messenger and regulates cellular functions,
has been identified as the major effector molecule involved in the
destruction of tumor cells by activated macrophages (Gordon &
Martinez, 2010; Lorsbach, Murphy, Lowenstein, Snyder & Russell,
1993). NO contributes to the killing of virally infected cells,
tumor cells and some pathogens remains unknown, partly because
it inactivates their mitochondrial respiratory chain enzymes. In
addition, a variety of cytokines that can regulate the cellular
and humoral immune responses were produced by activated
macrophages. TNF-␣ plays a crucial role in immune-regulation
and can also induce tumor cell apoptosis. Similarly, IL-1␤ is an
important cytokines. The cytokine from activated macrophages,
as a part of immune response, plays an important role in the
inhibition of cancer cells. Thus, the immunological activities of
OFPS11 was evaluated by monitoring its effects on the pro-
duction of NO and cytokines of RAW264.7 cells. As shown in
Table 1, NO, TNF-␣ and IL-1␤ induced by RAW264.7 cells indicated
3.5. Pinocytic activity assay
Since phagocytic activity is one of the most important func-
tions of macrophages in innate immune response (Zhao, Dong,
Chen & Hu, 2010), the effect of OFPS11 on phagocytic activities of
RAW264.7 cells was assessed by neutral red assay in this study. As
shown in Fig. 4C, OFPS11 appeared to enhance the phagocytic func-
tion of RAW264.7 in a prominent dose-related increment. Except
for the 62.5 ␮g/mL OFPS11 and 10 ␮g/mL LPS, the enhancement

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W. Zheng et al. / Carbohydrate Polymers 106 (2014) 335–342
Fig. 5. (A) iNOS protein expression in RAW264.7 cells treated with OFPS11. Adherent RAW264.7 cells were incubated with different concentrations of OFPS11 (0–1000 ␮g/mL)
and LPS (10 ␮g/mL) at 37 ◦C for 48 h. After incubation, the whole-cell lysates were prepared from the treated cells, and analyzed by western blot analysis as described in the
text. iNOS mRNA (B) and TNF-␣ mRNA (C) in RAW264.7 cells treated with OFPS11.Adherent RAW264.7 cells were incubated with OFPS11 (0, 62.5, 250, 1000 ␮g/mL) and
LPS (10 ␮g/mL) at 37 ◦C for 24 h. After incubation, iNOS and TNF-␣ mRNA levels in treated RAW264.7 cells were analyzed by RT-PCR method as described in the text. (D)
NF-␬B protein expression levels in RAW264.7 cells treated with OFPS11. Adherent RAW264.7 cells were incubated with different concentrations of OFPS11 (0–1000 ␮g/mL)
and LPS (10 ␮g/mL) at 37 ◦C. After 48 h, the nuclear lysates were prepared from the treated cells, and analyzed by western blot analysis as described in the text. OFPS11
(0–1000 ␮g/mL): C, O-62.5, O-125, O-250, O-500, O-1000; LPS: lipopolysaccharide.
significant dose-response increases in the secretion amount. The
difference in NO production between OFPS11 (500–1000 ␮g/mL)
and LPS (10 ␮g/mL) treated groups and normal control group were
statistically significant (ap < 0.05, aap < 0.01). This suggests that
OFPS11 may stimulate the NO production of RAW264.7 cells. In
addition, OFPS11 and LPS treated-RAW264.7 cells secreted more
TNF-␣ and IL-1␤ than that in the normal control group (ap < 0.05,
aap < 0.01). These results suggest that OFPS11 shows beneficial
effects on enhancing the production of NO and the secretion of
cytokines.
Table 1
Effect of OFPS11 on inducing NO, TNF-␣ and IL-1␤ production of RAW264.7 cells.
Groups
NO levels
(␮mol/mL)
TNF-␣ levels
(pg/mL)
IL-1␤ levels
(pg/mL)
Normal control group
OFPS11 (62.5 ␮g/mL)
OFPS11 (125 ␮g/mL)
OFPS11 (250 ␮g/mL)
OFPS11 (500 ␮g/mL)
OFPS11 (1000 ␮g/mL)
LPS (10 ␮g/mL)
2.79 0.18
3.07 0.15
3.22 0.08
3.33 0.65
4.87 0.25a
10.37 0.98aa
11.83 0.79aa
200.03 7.18
207.39 5.83
225.51 7.65a
268.09 6.59aa
344.96 4.70aa
436.93 4.86aa
348.04 2.25aa
65.96 2.07
80.27 3.18a
123.58 2.36aa
130.92 3.98aa
147.60 4.16aa
150.77 2.03aa
122.78 1.68aa
RAW 264.7 Cells (2 × 104 cells/well) were stimulated with OFPS11 (0–1000 ␮g/mL)
and LPS (10 ␮g/mL) for 24 h. The supernatant NO, TNF-␣ and IL-1␤ levels were
determined using commercial test kit. Each value is presented as mean SD of 5 sep-
arate experiments. P values are shown as ap < 0.05, aap < 0.01 compared with normal
control group. A value of p < 0.05 denotes the presence of a statistically significant
difference according to Tukey method.
3.7. Effects of OFPS11 on protein expression of iNOS and mRNA
expressions of iNOS and TNF-˛ in RAW264.7 cells
iNOS is a significant isoform of enzyme nitric oxide synthase
(NOS) which induces the synthesis of NO (Davis, Martin, Turko

W. Zheng et al. / Carbohydrate Polymers 106 (2014) 335–342
341
& Murad, 2001). In addition, the published data showed that
4. Conclusion
polysaccharide induce NO production from RAW264.7 cells by
up-regulating the expression of iNOS protein and iNOS Mrna
(Nakamura, Suzuki, Wada, Kodama & Doi, 2006). Thus, the possi-
ble mechanism involved in the up-regulating of NO production was
examined in the protein and mRNA expressions of OFPS11-treated
RAW264.7.
OFPS11 is a water-soluble polysaccharide mainly composed of
galactose and rhamnose in a molar ratio of 2.23:1 with molecu-
lar mass of 1700 kDa. The results revealed that OFPS11 exhibited
an indirect cytotoxic effect on HepG-2 cancer cells by activating
RAW264.7 cells. Meanwhile, OFPS11 could promote the phago-
cytosis, production of NO and cytokines of RAW264.7 cells when
comparing with normal control groups. In addition, the expressions
of iNOS in both protein and mRNA level, TNF-␣ in mRNA level and
NF-␬B in protein level were enhanced in OFPS11-treated groups.
Thus, this study provides evidence of plant-derived polysaccharide
OFPS11 with non-toxic nature, which could be used as a poten-
tial antitumor immunomodulatory agent used in health food and
pharmaceutical therapy. Since activities of polysaccharides depend
on their chemical structure, the future work will be conducted
to determine the concrete structure of OFPS11 and its targets in
macrophages.
As shown in Fig. 5A, western blot analysis revealed that lev-
els of protein were significantly expressed in OFPS11-treated and
LPS-stimulated RAW264.7 cells. However, in normal control group,
macrophage cells showed very low levels of iNOS protein, which
indicates that OFPS11 could significantly stimulate the expression
of iNOS protein of macrophages. Consistent with the NO pro-
duction and iNOS protein expression, RT-PCR analysis exhibited
higher iNOS mRNA expression in OFPS11-induced group than that
in normal control group, though the expressions were not in a
dose-related way (Fig. 5B). Furthermore, the LPS (10 ␮g/mL) could
up-regulate the expression of iNOS mRNA significantly. The results
suggested that the increasing of NO levels in activated macrophages
might be due to the enhancement of protein and mRNA expres-
sion of iNOS. The enhanced expression of mRNA level of TNF-␣ was
also observed from Fig. 5C, after treatment with various doses of
OFPS11 (62.5, 250 and 1000 ␮g/mL) and LPS (10 ␮g/mL) for 24 h, the
higher concentrations of OFPS11 (250 and 1000 ␮g/mL) enhanced
expression of mRNA markedly comparing with normal control
group, though all the expressions were almost trace level. Consis-
tent with the increase of TNF-␣ secretion, this observation suggests
that OFPS11 could enhance TNF-␣ production by up-regulated
TNF-␣ gene expression. Since gene expressions are regulated by
mediators, the results may indicate that OFPS11 increased the NO
production and TNF-␣ by a mechanism involving transcriptional
regulations.
Acknowledgements
This work was supported financially by Natural Science Fund of
Jiangsu Province (BK2012702). We also appreciate Dr. Samuel Jerry
Cobbina for language polishing.
References
Bai, L., Zhu, L. Y., Yang, B. S., Shi, L. J., Liu, Y., Jiang, A. M., et al. (2012). Antitumor
and immunomodulating activity of a polysaccharide from Sophora flavescens
Ait. International Journal of Biological Macromolecules, 51, 705–709.
Bontà, I. L., & Ben-Efraim, S. (1993). Involvement of inflammatory mediators in
macrophage antitumor activity. Journal of Leukocyte Biology, 54(6), 613–626.
Cai, Z., Li, W., Wang, H., Yan, W., Zhou, Y., Wang, G., et al. (2012a). Anti-tumor and
immunomodulating activities of a polysaccharide from the root of Sanguisorba
officinalis L. International Journal of Biological Macromolecules, 51(4), 484–488.
Cai, Z., Li, W., Wang, H., Yan, W., Zhou, Y., Wang, G., et al. (2012b). Antitumor effects
of a purified polysaccharide from Rhodiola rosea and its action mechanism. Car-
bohydrate Polymers, 90(1), 296–300.
3.8. The protein expression of NF-ꢀB
Previous study suggested that polysaccharide induced NO and
cytokines production from the RAW264.7 cells through the acti-
vation of transcription factor NF-␬B (Lee et al., 2004). NF-␬B
usually stays quietly as an inactive, non-covalent binding form
with p50-p65-I␬B trimer in the cytoplasm. When signals for the
activation of NF-␬B are received, serine residues in I␬B are phos-
phorylated and dissociated from NF-␬B, which are then transferred
to the nucleus as an activated transcription factor (Ley, 2004).
After the degradation of I␬B, the binding sites of p50–p65 dimer
are then exposed to combine with the ␬B motif. Then the NF-
␬B p65 subunit exhibits a potent activity in regulating many
important biological and pathological processes and modulating
the transcription of a large number of genes involving iNOS, IL-
1␤ and TNF-a. Thus, NF-␬B p65 in nuclear was examined using
Western-blotting.
The outcome shown in Fig. 5D suggested that NF-␬B was
tremendously activated in OFPS11-treated RAW264.7 cells when
comparing with normal control group, and the extent of the activa-
tion were in an evidently concentration-dependent manner, which
indicated that NF-␬B activation was required for OFPS11-induced
macrophage activation. Thus, the results demonstrated that NF-
␬B p65 in nuclear was significantly increased after stimulation
with OFPS11, which suggests that OFPS11 can stimulate the release
of NF-␬B from I␬B and transfer from cytoplasm to cytoplast. In
summary, the results suggested that OFPS11 could potentially be
used as an immune-modulatory agent, and it induced antitumor
responses by activated RAW264.7 macrophages, at least in part,
by up-regulating the expressions of NO and TNF-␣ through NF-␬B
signaling pathway.
Cao, X. F., Yuan, T., Sun, S., & Sun, R. C. (2011). Isolation and characterization of
soluble polysaccharides from Calamagrostis angustifolia Kom. Bioresources, 6(3),
2896–2911.
Davis, K. L., Martin, E., Turko, I. V., & Murad, F. (2001). Novel effects of nitric oxide.
Annual Review of Pharmacology and Toxicology, 41(1), 203–236.
Di, H., Zhang, Y., & Chen, D. (2013). An anti-complementary polysaccharide from the
roots of Bupleurum Chinense. International Journal of Biological Macromolecules,
58, 179–185.
Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P., & Smith, F. t. (1956). Colorimetric
method for determination of sugars and related substances. Analytical Chemistry,
28(3), 350–356.
Fan, Y., Wu, X., Zhang, M., Zhao, T., Zhou, Y., Han, L., et al. (2011). Physical char-
acteristics and antioxidant effect of polysaccharides extracted by boiling water
and enzymolysis from Grifola frondosa. International Journal of Biological Macro-
molecules, 48(5), 798–803.
Gordon, S., & Martinez, F. O. (2010). Alternative activation of macrophages: Mecha-
nism and functions. Immunity, 32(5), 593–604.
Han, S.-B., Lee, C. W., Kang, M. R., Yoon, Y. D., Kang, J. S., Lee, K. H., et al. (2006).
Pectic polysaccharide isolated from Angelica gigas Nakai inhibits melanoma cell
metastasis and growth by directly preventing cell adhesion and activating host
immune functions. Cancer Letters, 243(2), 264–273.
Hromádková, Z., Paulsen, B. S., Polovka, M., Kosˇt’álová, Z., & Ebringerová, A. (2013).
Structural features of two heteroxylan polysaccharide fractions from wheat
bran with anti-complementary and antioxidant activities. Carbohydrate Poly-
mers, 93(1), 22–30.
Jarret, R. L., Wang, M. L., & Levy, I. J. (2011). Seed oil and fatty acid content in okra
(Abelmoschus esculentus) and related species. Journal of Agricultural and Food
Chemistry, 59, 4019–4024.
Jiang, H., Wang, J., Zhou, A., Xie, G., Yan, L., Jiang, Y., et al. (2013). Purification, charac-
terization and antiviral activity of two heteropolysaccharides from Duchesneae
Indicae. International Journal of Biological Macromolecules, 58, 287–295.
Jin, X. (2012). Bioactivities of water-soluble polysaccharides from fruit shell of
Camellia oleifera Abel: Antitumor and antioxidant activities. Carbohydrate Poly-
mers, 87(3), 2198–2201.
Kim, H. S., Kim, Y. J., Lee, H. K., Ryu, H. S., Kim, J. S., Yoon, M. J., et al. (2012). Activation
of macrophages by polysaccharide isolated from Paecilomyces cicadae through
toll-like receptor 4. Food and Chemical Toxicology, 50(9), 3190–3197.

342
W. Zheng et al. / Carbohydrate Polymers 106 (2014) 335–342
Kim, J. Y., Yoon, Y. D., Ahn, J. M., Kang, J. S., Park, S.-K., Lee, K., et al. (2007).
Angelan isolated from Angelica gigas Nakai induces dendritic cell matura-
tion through toll-like receptor 4. International Immunopharmacology, 7(1),
78–87.
Klimp, A., De Vries, E., Scherphof, G., & Daemen, T. (2002). A potential role of
macrophage activation in the treatment of cancer. Critical Reviews in Oncol-
ogy/Hematology, 44(2), 143–161.
López-Barajas, M., López-Tamames, E., & Buxaderas, S. (1998). Improved size-
exclusion high-performance liquid chromatographic method for the simple
analysis of grape juice and wine polysaccharides. Journal of Chromatography A,
823(1), 339–347.
Lee, K. Y., You, H. J., Jeong, H. G., Kang, J. S., Kim, H. M., Rhee, S. D., et al. (2004). Polysac-
charide isolated from Poria cocos sclerotium induces NF-(B/Rel activation and
iNOS expression through the activation of p38 kinase in murine macrophages.
International Immunopharmacology, 4(8), 1029–1038.
Shen, H., Tang, G., Zeng, G., Bi, Y., Yang, Y., Cai, X., et al. (2012). Purification and
characterization of an antitumor polysaccharide from Portulaca oleracea L. Car-
bohydrate Polymers, 93, 395–400.
Sheu, S.-C., & Lai, M.-H. (2012). Composition analysis and immuno-modulatory effect
of okra (Abelmoschus esculentus L.) extract. Food Chemistry, 134(4), 1906–1911.
Shi, B. J., Nie, X. H., Chen, L. Z., Liu, Y. L., & Tao, W. Y. (2007). Anticancer activities of a
chemically sulfated polysaccharide obtained from Grifola frondosa and its combi-
nation with 5-fluorouracil against human gastric carcinoma cells. Carbohydrate
Polymers, 68(4), 687–692.
Wang, M., Jiang, C., Ma, L., Zhang, Z., Cao, L., Liu, J., et al. (2013). Preparation, pre-
liminary characterization and immunostimulatory activity of polysaccharide
fractions from the peduncles of Hovenia dulcis. Food Chemistry, 138(1), 41–47.
Wu, W., Zhu, Y., Zhang, L., Yang, R., & Zhou, Y. (2012). Extraction, preliminary struc-
tural characterization, and antioxidant activities of polysaccharides from Salvia
miltiorrhiza Bunge. Carbohydrate Polymers, 87(2), 1348–1353.
Lengsfeld, C., Titgemeyer, F., Faller, G., & Hensel, A. (2004). Glycosylated compounds
from okra inhibit adhesion of Helicobacter pylori to human gastric mucosa. Jour-
nal of Agricultural and Food Chemistry, 52(6), 1495–1503.
Wu, X., Mao, G., Fan, Q., Zhao, T., Zhao, J., Li, F., et al. (2012). Isolation, purifica-
tion, immunological and anti-tumor activities of polysaccharides from Gymnema
sylvestre. Food Research International, 48(2), 935–939.
Ley, S. B. S. C. (2004). Functions of NF-␬B1 and NF-␬B2 in immune cell biology.
Biochemical Society, 382, 393–409.
Liao, C. H., & Lin, J. Y. (2013). Purified active lotus plumule (Nelumbo nucifera Gaertn)
polysaccharides exert anti-inflammatory activity through decreasing toll-like
receptor-2 and -4 expressions using mouse primary splenocytes. Journal of
Ethnopharmacology, 147, 164–173.
Lorsbach, R. B., Murphy, W. J., Lowenstein, C. J., Snyder, S. H., & Russell, S. (1993).
Expression of the nitric oxide synthase gene in mouse macrophages activated
for tumor cell killing. Molecular basis for the synergy between interferon-
gamma and lipopolysaccharide. Journal of Biological Chemistry, 268(3),
1908–1913.
Mao, G., Zou, Y., Feng, W., Wang, W., Zhao, T., Ye, C., et al. (2014). Extraction,
preliminary characterization and antioxidant activity of Se-enriched Maitake
polysaccharide. Carbohydrate Polymers, 101, 213–219.
Xu, C. P., Xiao, Y., & Mao, D. B. (2013). Antioxidant activity potential of virginia (Flue-
Cured) tobacco flower polysaccharide fractions obtained by ultrasound-assisted
extraction. Bioscience Biotechnology, and Biochemistry, 77(10), 2100–2104.
Yang, L. C., Hsieh, C. C., Lu, T. J.,
& Lin, W. C. (2013). Structurally char-
acterized arabinogalactan from Anoectochilus formosanus as an immuno-
modulator against CT26 colon cancer in BALB/c mice. Phytomedicine,
http://dx.doi.org/10.1016/j.phymed.2013.10.032
Yang, L., Zhao, T., Wei, H., Zhang, M., Zou, Y., Mao, G., et al. (2011). Carboxymethyla-
tion of polysaccharides from Auricularia auricula and their antioxidant activities
in vitro. International Journal of Biological Macromolecules, 49(5), 1124–1130.
Zhang, C. X., & Dai, Z. R. (2011). Immunomodulatory activities on macrophage of a
polysaccharide from Sipunculus nudus L. Food and Chemical Toxicology, 49(11),
2961–2967.
Zhang, J., Liu, Y. j, Park, H. s., Xia, Y. m., & Kim, G. s. (2012). Antitumor activity of sul-
fated extracellular polysaccharides of Ganoderma lucidum from the submerged
fermentation broth. Carbohydrate Polymers, 87(2), 1539–1544.
Zhao, L., Dong, Y., Chen, G., & Hu, Q. (2010). Extraction, purification, characterization
and antitumor activity of polysaccharides from Ganoderma lucidum. Carbohy-
drate Polymers, 80(3), 783–789.
Zhao, Q., Xie, B., Yan, J., Zhao, F., Xiao, J., Yao, L., et al. (2012). In vitro antioxidant
and antitumor activities of polysaccharides extracted from Asparagus officinalis.
Carbohydrate Polymers, 87(1), 392–396.
Molfetta, I., Ceccarini, L., Macchia, M., Flamini, G., & Cioni, P. L. (2013). Abelmoschus
esculentus (L.) Moench. and Abelmoschus moschatus Medik: Seeds production
and analysis of the volatile compounds. Food Chemistry, 141, 34–40.
Nakamura, T., Suzuki, H., Wada, Y., Kodama, T.,
& Doi, T. (2006). Fucoidan
induces nitric oxide production via p38 mitogen-activated protein kinase
and NF-kappaB-dependent signaling pathways through macrophage scav-
enger receptors. Biochemical and Biophysical Research Communications, 343(1),
286–294.
Oliveira, A. J. B. d., Cordeiro, L., Gonc¸ alves, R. A. C., Ceole, L. F., Ueda-Nakamura,
T., & Iacomini, M. (2013). Structure and antiviral activity of arabinogalactan
with (1 → 6)-␤-d-galactan core from Stevia rebaudiana leaves. Carbohydrate
Polymers, 94, 179–184.
Zhao, T., Mao, G., Mao, R., Zou, Y., Zheng, D., Feng, W., et al. (2013). Antitumor and
immunomodulatory activity of a water-soluble low molecular weight polysac-
charide from Schisandra chinensis (Turcz.) Baill. Food and Chemical Toxicology, 55,
609–616.
Shang, P., Qian, A. R., Yang, T. H., Jia, M., Mei, Q. B., Cho, C. H., et al. (2003). Experimen-
tal study of anti-tumor effects of polysaccharides from Angelica sinensis. World
Journal of Gastroenterology, 9(9), 1963–1967.
Zhao, T., Mao, G., Zhang, M., Zou, Y., Feng, W., Gu, X., et al. (2014). Enhanced antitumor
and reduced toxicity effect of Schisanreae polysaccharide in 5-Fu treated Heps-
bearing mice. International Journal of Biological Macromolecules, 63, 114–118.