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Enhanced immunostimulatory and antitumor activity
of different derivatives of κ-carrageenan oligosaccharides
from Kappaphycus striatum
Huamao Yuan & Jinming Song & Xuegang Li & Ning Li &
Song Liu
Received: 24 January 2010 / Revised and accepted: 12 May 2010 /Published online: 12 June 2010
# Springer Science+Business Media B.V. 2010
Abstract Chemical modification of carbohydrates can lead
to differences in their biological activities. We previously
showed that κ-carrageenan oligosaccharides from Kappa-
phycus striatum have antitumor and immunomodulation
effects on S180-bearing mice. In this study, we tested the
hypothesis that different chemical modifications of carra-
geenan oligosaccharides enhance their activities. The mice
inoculated with S180 cell suspension were treated p.o. with
carrageenan oligosaccharides and their sulfated, acetylated,
and phosphorylated derivatives (50, 100, and 200 μg g−1)
for 14 days. Transplantable tumor inhibition rate and
macrophage phagocytosis, quantitative hemolysis of sheep
red blood cells, lymphocyte proliferation, the activity of
natural killer cells, production of interleukin-2, and tumor
necrosis factor-α were also analyzed. As expected, treat-
ment with different κ-carrageenan oligosaccharides deriva-
tives resulted in an increase in tumor inhibition rate and
macrophage phagocytosis and cellular immunity, especially
on spleen lymphocyte proliferation. The sulfated derivative
at the dose 200 μg g−1 per day showed the highest
antitumor activity with the 54.12% tumor weight inhibition
and elicited an increase in nature killer cells activity up to
76.1% on S180-bearing mice, which were both significant-
ly higher than the unmodified oligosaccharides. It sug-
gested that chemical modification (especially sulfation) of
carrageenan oligosaccharides can enhance their antitumor
effect and boost their antitumor immunity.
Keywords Carrageenan . Oligosaccharides . Derivatives .
Antitumor . Immunomodulation . Chemical modification
Introduction
Tumors develop a wide variety of immunosuppressive
strategies to escape from immune responses. These mech-
anisms include downregulation of target antigens and
antigen-presenting machinery, secretion of inhibitory cyto-
kines, and recruitment of regulatory immune cells to the
tumor site (Rabinovich et al. 2007). Even worse, traditional
chemotherapeutic drugs such as cyclophosphamide, which
are used to kill tumor cells, have a suppressive effect on the
immune system at the mean time. Complex oligosacchar-
ides have been thought of as a food source for a healthy
diet, and their biological roles were limited to antigenic
properties of various blood groups (Weymouth-Wilson
1997). In recent years, various compounds, either related
to carbohydrates or carbohydrate itself, have been recog-
nized as important biologically active compounds (Saksena
et al. 1999). Many organisms such as marine algae contain
sulfated polysaccharides that are vital for their biological
activity, and carbohydrates have key roles in a broad range
of biological processes, including signal transduction and
immune responses. Oligosaccharides have been shown to
have a variety of effects on the immune system, such as
inhibition of cancer metastasis, antitumor activity, immu-
nological activity, and complement activation (Bland et
al. 2004), and may be effective candidates for tumor
immunotherapy.
The bioactivity of sulfated polysaccharides/oligosacchar-
ides depends on several structural features such as the
degree of sulfation (DS), the molecular weight, the sulfation
position, type of sugar, and glycosidic branching. Chemical
modification of the carbohydrates can lead to differences in
their biological activities. Chemical oversulfation of fucoi-
dan can enhance its antiangiogenic and antitumor activities
(Soeda et al. 2000; Koyanagi et al. 2003). A polysaccharide
H. Yuan : J. Song (*) :X. Li :N. Li : S. Liu
Institute of Oceanology, Chinese Academy of Sciences,
Qingdao 266071, People’s Republic of China
e-mail: jmsong@ms.qdio.ac.cn
J Appl Phycol (2011) 23:59–65
DOI 10.1007/s10811-010-9536-4
fraction with a high sulfate content from Porphyra
yezoensis possesses the strongest macrophage stimulating
activity among the polysaccharide fractions, which suggests
that a high amount of sulfate groups contributes to the
activity (Yashizawa et al. 1995). In our previous work, κ-
carrageenan oligosaccharides from Kappaphycus striatum
were found to have antitumor activity both in vitro and in
vivo and immunomodulation effects on S180-bearing mice
(Yuan and Song 2005; Yuan et al. 2006a). We also prepared
sulfated, acetylated, and phosphorylated derivatives of κ-
carrageenan oligosaccharides and found that the different
derivatives exhibited higher antioxidant activity than
carrageenan oligosaccharides in certain antioxidant systems
in vitro, which indicated that the chemical modification of
carrageenan oligosaccharides can enhance their antioxidant
activity (Yuan et al. 2005; Yuan et al. 2006b). In the present
study, the antitumor and immunomodulative effects of
different κ-carrageenan oligosaccharides derivatives on
S180-bearing mice were investigated in order to evaluate
whether the chemical modification will have any influence
on the activities.
Materials and methods
Carrageenan was purchased from Yantai Algae Industries
(Shandong, China). The carrageenan was treated with
NaOH and KCl to increase the content of An residues to
enhance gel strength. Properties provided by the manufac-
turer include gel strength of 800 g cm−2 at 1.5% water and
gel point, 41°C. The carrageenan polysaccharide was
degraded by mild acidic hydrolysis to obtain the carrageen-
an oligosaccharide mixture described previously (Yuan and
Song 2005). Carrageenan oligosaccharides and their sulfat-
ed, acetylated, and phosphorylated derivatives were pre-
pared in our laboratory, with average molecular weights
estimated to be 1.2, 0.8, 1.2, and 1.1 kDa, respectively
(Yuan et al. 2005). The sulfate content of oligosaccharides
and sulfated derivative is 8.98% and 47.9% (w/w), respec-
tively. The degree of acetylation (DSAC) of the acetylated
derivative is 1.13, and the total phosphate content of the
phosphorylated derivative is 2.98% (w/w). MTT and ConA
were purchased from Sigma/Aldrich Chemical Co. Cytokine
levels were determined by enzyme-linked immunosorbent
assays (ELISA) using the reagents and protocol provided by
the manufacturer (Jingmei Biotech Co., Ltd, Shanghai,
China) for interleukin-2 (IL-2) and tumor necrosis factor-α
(TNF-α). All other reagents were of analytical grade.
Male BALB/c mice (20±2 g, 6–8 weeks old) were
purchased from the Animal Center, Medical Institute of
Shandong University, China. The mice were housed under
normal laboratory conditions (21±2°C, 12/12-h light–dark
cycle) with free access to standard rodent chow and water.
Under sterile condition, 0.2 mL of S180 cell suspension
(about 3×106cells mL−1) was subcutaneously inoculated
into the mouse armpit. The inoculated mice were divided
randomly into S180-bearing control group, 120 μg g−1
ftorafur (FT207) and 50, 100, and 200 μg g−1 carrageenan
oligosaccharides and their different derivatives groups. A
normal control group was also used in this experiment (ten
mice in each group). FT207, oligosaccharides, and their
derivatives were administrated p.o. for 14 days once daily.
Normal control and S180-bearing control groups received
the same volume of distilled water. On day15, all animals
were executed. The mice, thymus, spleen, and tumors were
weighed, and a count on tumor inhibition rate was made.
Thymus and spleen indexes are expressed as the thymus
and spleen weight relative to body weight. Among the ten
mice in each group, six mice (randomly selected) were used
for lymphocyte proliferation and natural killer (NK) cell
activity assay. For macrophage phagocytosis, quantitative
hemolysis of sheep red blood cells (QHS), and cytokines
assay, mice were divided into three groups, and each group
also divided into 15 sub-groups(six mice in each sub-
group): i.e., normal group, S180-bearing control group,
120 μg g−1 ftorafur (FT207), and 50, 100, and 200 μg g−1
carrageenan oligosaccharides and their different derivatives
groups.
Macrophage phagocytosis assay (carbon clearance)
Carbon clearance was assessed according to the method of
Salem et al. (1999) with some modifications. Carbon ink
was diluted to 1.6 g mL−1 in phosphate-buffered saline
(PBS) supplemented with 10% gelatin. Fourteen days after
the carrageenan oligosaccharides and their different deriv-
atives treatment, each mouse was i.v. injected with the
diluted carbon ink at a dose of 0.01 mL g−1 body weight.
At 3, 6, 9, 12, and 15 min after the injection, blood was
obtained by retro-orbital venous puncture, and 0.02 mL of
blood was added to 2 mL of 0.1% Na2CO3. Then, the
optical density (OD) of the samples was determined
spectrophotometrically at 620 nm. The clearance value K
was calculated according to the following formula:
K ¼ logOD1 � logOD2ð Þ= t2 � t1ð Þ
where OD1 and OD2 represent the value of OD at the time
t1 and t2.
Lymphocyte proliferation assay
Spleens were aseptically removed from sacrificed mice with
scissors and forceps in cold PBS, gently homogenized with
a loose teflon pestle, and passed through a sterilized mesh
(200 mesh) to obtain single-cell suspensions. Erythrocytes
in the cell mixture were washed by the hypoosmotic
60 J Appl Phycol (2011) 23:59–65
hemolysis rapidly. Finally, the cells were suspended to a
final density of 5×106cells mL−1 in RPMI-1640 medium
supplemented with 10% newborn bovine serum. Spleen
cells (200 μL well−1) were seeded into a 96-well plate in
the presence of ConA (8 μg mL−1) and cultured at 37°C in
5% CO2 atm. After incubation for 72 h, 10 μL MTT
(5 mg mL−1) was added to each well, and the plate was
incubated for another 4 h. The plate was then centrifuged at
2,000 rpm for 10 min, and the supernatants were discarded.
A total of 200 μL DMSO was added to each well and
shaken until crystals were dissolved. The absorbance A570
was detected on a Microplate Reader (Bio-Rad, USA).
Quantitative hemolysis of sheep red blood cells
(QHS) assay
QHS assay was performed using the method of Simpson
and Gozzo (1978) with some modifications. In brief,
0.2 mL of 5% sheep red blood cells (SRBC) prepared in
normal saline was injected to animals, i.p., 6 days prior to
the assay. On day6 following immunization, the spleens
were removed, and single-cell suspensions of 5×106cells/
mL mL−1 were prepared in PBS. A total of 1.0 mL 0.2%
SRBC and 1.0 mL of guinea pig serum were mixed with
1.0 mL of cell suspension and incubated for 1 h at 37°C.
After centrifugation at 3,000 rpm for 5 min, the absorbance
of the supernatant was measured at 413 nm.
Assay of natural killer (NK) cells activity
YAC-1 cells were used as target cells and seeded in 96-well
U-bottom culture plates at 1×104cells well−1 in RPMI-
1640. Spleen cells prepared as described above were used
as the effector cells and were added at 5×105cells well−1 to
give an E/T ratio of 50:1. The plates were then incubated for
20 h at 37°C in 5% CO2 atm. Of the supernatant, 100 μL
was collected from each well, and 10 μL MTT (5 mg mL−1)
was added to each well, and the plate was incubated for
another 4 h and subjected to MTT cellular assay. Three kinds
of control measurements were performed: target cells
control, blank control, and effector cells control. NK cell
activity was calculated as the following equation:
NK cell activity %ð Þ ¼ ODT � ODS � ODEð Þ½ �=ODT � 100%;
where ODT=optical density value of target cells control,
ODS=optical density value of test samples, and ODE=
optical density value of effector cells control.
Cytokine assay
Serum was collected by retro-orbital venous puncture 24 h
after the last administration of the oligosaccharides and
their different derivatives. Two-site sandwich ELISA were
performed to quantify IL-2 and TNF-α in the serum
according to the manufacturer’s instructions. IL-2 and
TNF-α concentrations were calculated from the absorbance
values by plotting the values against IL-2 and TNF-α
standard curves, which were performed for each assay.
Statistical analysis
Results are expressed as mean±standard deviation (SD).
The statistical significance of the differences between groups
was evaluated by the analysis of variance, followed by
Student’s t test. Significant differences were set at P<0.05.
Results
Effects of carrageenan oligosaccharides derivatives
on thymus, spleen indexes, and tumor weights
As shown in Table 1, all the carrageenan oligosaccharides
and their derivatives could significantly inhibit the growth
of mouse transplanted sarcoma S180 except the oligosac-
charides at the low dose at 50 μg g−1. Furthermore, all the
chemically modified carrageenan oligosaccharides showed
enhanced inhibitory effects compared to carrageenan
oligosaccharides at the same dose level, but there are no
significant differences between them except the sulfated
derivative at the dose 200 μg g−1. The inhibition rates of
sulfated and phosphorylated derivatives are similar to the
chemotherapeutic drug FT207 (120 μg g−1) at the dose
100 μg g−1. All the oligosaccharide-treated groups had a
significant increase in the thymus and spleen indexes
compared with the S180 control group, while FT207 did
not increase the thymus index and even decreased the
spleen index.
Effects of carrageenan oligosaccharides derivatives
on macrophage phagocytosis and humoral immunity
Carrageenan oligosaccharides and their different derivatives
markedly increased macrophage phagocytosis in S180-
bearing mice (Table 2). The sulfated and phosphorylated
derivatives at a dose of 200 μg g−1 showed higher
phagocytic activity than other samples, but there was no
significant elevation of phagocytosis by all derivatives
compared to carrageenan oligosaccharides. Carrageenan
oligosaccharides at all doses, and only the derivatives at
high dose, significantly increased the production of antibody
secreted by spleen cells in S180-bearing mice. The effect of
carrageenan oligosaccharides at a dose of 200 μg g−1 was
the best and could restore humoral immunity in S180-
bearing mice to the level of the normal control. The positive
J Appl Phycol (2011) 23:59–65 61
control FT207, however, effectively decreased phagocytic
activity and humoral antibody production.
Effects of carrageenan oligosaccharides derivatives
on cellular immunity
In order to understand the immunomodulatory activity of
carrageenan oligosaccharides and their derivatives, we
investigated their effects on the proliferation of splenic
cells. With the exception of the acetylated derivative at low
dose, all other derivatives significantly increased the
proliferation of splenic cells in a dose-dependent manner
(Table 3). Furthermore, sulfated derivative at all doses,
acetylated and phosphorylated derivatives at medium and
high dose, significantly enhanced the proliferation of
splenic cells compared to carrageenan oligosaccharides at
the same dose level.
Tumor cell elimination is known to be mediated in part
by the cytotoxic activity of NK cells. We therefore
measured the cytotoxic activity of splenocytes against
NK-sensitive (YAC-1) tumor cells. Carrageenan oligosac-
charides and their derivatives increased NK cell activity in
a dose-depended manner, but there were no significant
differences among the groups except for the high dose of
carrageenan oligosaccharides and their sulfated and phos-
phorylated derivatives and the medium dose of sulfated
derivatives. In addition, the sulfated derivative also signif-
icantly increased NK activity compared to carrageenan
oligosaccharides at high dose and even exceeded that of the
normal mice.
Effects of carrageenan oligosaccharides derivatives
on cytokine production in serum
The effect of carrageenan oligosaccharides and their de-
rivatives on IL-2 and TNF-α level in murine serum were
determined by ELISA. As seen in Table 4, the production of
IL-2 and TNF-α were significantly decreased in S180-bearing mice compared with the normal control. Carrageen-
an oligosaccharides markedly augmented IL-2 production in
serum of S180-bearing mice in a dose-depended manner.
They also increased TNF-α production in a dose-dependent
manner and could stimulate TNF-α production to normal
control in serum of S180-bearing mice at high dose. How-
ever, the chemical modification of the carrageenan oligosac-
charides did not enhance and even decreased the production
of cytokines in some cases in the serum of S180-bearing
mice.
Table 1 Effects of carrageenan oligosaccharides and their derivatives on thymus, spleen indexes, and tumor weights in S180-bearing mice
Group Dose
(μg/g−1 day−1)
Thymus index
(mg g−1)
Spleen index
(mg g−1)
Tumor
weight (g)
Inhibition
rate (%)
Normal control – n.d. n.d. – –
S180 control – 1.46±0.43 6.69±2.50 1.83±0.55 –
FT207 120 1.65±1.02 5.22±1.09 0.98±0.33** 46.43
Carrageenan oligosaccharides 50 3.50±1.41* 10.79±4.61* 1.32±0.44 27.81
100 3.95±1.14** 11.95±6.01* 1.17±0.23** 36.30
200 4.39±1.32*** 12.78±2.67*** 1.10±0.21** 39.80
Sulfated derivative 50 3.81±1.67* 14.65±6.26** 1.01±0.33** 44.66
100 4.31±0.83*** 16.69±5.90*** 1.03±0.18** 43.80
200 5.55±2.72** 13.85±4.74*** 0.92±0.32*** 54.12****
Acetylated derivative 50 2.93±1.10 12.26±4.10** 1.03±0.13* 43.79
100 4.12±1.68** 12.85±5.40** 1.11±0.30* 39.38
200 4.43±1.46** 14.80±4.99*** 0.91±0.16** 50.17
Phosphorylated derivative 50 3.11±1.28 12.46±4.56** 1.23±0.29* 33.02
100 4.29±1.52** 13.97±3.66*** 1.00±0.27*** 45.49
200 4.60±0.84*** 12.78±3.97** 0.90±0.31*** 51.03
S180-bearing mice were administered p.o. with FT207 120 μg g−1 and carrageenan oligosaccharides and their derivatives 50, 100, and 200 μg g−1 for
14 days once daily. S180-bearing control group received the same volume of distilled water. Thymus, spleen indexes, tumor weight, and the inhibitory rate
were determined on day15. Values are mean±SD of ten mice
n.d. not determined
*Significantly different from S180 control group at P<0.05
**Significantly different from S180 control group at P<0.01
***Significantly different from S180 control group at P<0.001
****Significantly different from corresponding the same dose level of carrageenan oligosaccharides group at P<0.05
62 J Appl Phycol (2011) 23:59–65
Table 3 Effects of carrageenan oligosaccharides and their derivatives on spleen lymphocyte proliferation and NK cells activity in S180-bearing mice
Group Dose (μg g−1 day−1) Lymphocyte proliferation A570 NK activity (%)
Normal control – 0.412±0.020** 56.6±3.2**
S180 control – 0.277±0.048 15.8±8.8
FT207 120 0.228±0.056 9.8±5.3
Carrageenan oligosaccharides 50 0.338±0.021 16.5±6.9
100 0.340±0.021 36.9±17.8
200 0.345±0.016 53.0±6.0**
Sulfated derivative 50 0.397±0.015**,**** 16.0±12.0
100 0.430±0.033**,*** 51.5±12.4*
200 0.438±0.029*,**** 76.1±13.7**,***
Acetylated derivative 50 0.330±0.027 12.3±2.5
100 0.415±0.039**,*** 25.5±15.0
200 0.425±0.035**,*** 31.0±10.4
Phosphorylated derivative 50 0.363±0.022* 19.9±14.4
100 0.457±0.072**,*** 41.9±14.0
200 0.471±0.068**,*** 61.0±6.8**
S180-bearing mice were administered p.o. with FT207 120 μg g−1 and carrageenan oligosaccharides and their derivatives 50, 100, and 200 μg g−1 for
14 days once daily. Normal control and S180-bearing control group received the same volume of distilled water. Values are mean±SD of six mice
*Significantly different from S180 control group at P<0.05
**Significantly different from S180 control group at P<0.01
***Significantly different from corresponding the same dose level in carrageenan oligosaccharides group at P<0.05
****Significantly different from corresponding the same dose level in carrageenan oligosaccharides group at P<0.01
Table 2 Effects of carrageenan oligosaccharides and their derivatives on phagocytosis and the form of antibody secreted by spleen cells in S180-
bearing mice
Group Dose (μg g−1 day−1) K A413
Normal control – n.d. 0.852±0.191**
S180 control – 0.026±0.005 0.467±0.044
FT207 120 0.017±0.003** 0.384±0.051*
Carrageenan oligosaccharides 50 0.044±0.005*** 0.787±0.159**
100 0.044±0.009*** 0.797±0.106**
200 0.042±0.009** 0.853±0.138**
Sulfated derivative 50 0.041±0.008** 0.575±0.120
100 0.041±0.008** 0.638±0.098
200 0.053±0.029* 0.718±0.116**
Acetylated derivative 50 0.042±0.010** 0.633±0.181
100 0.053±0.015*** 0.572±0.187
200 0.048±0.008*** 0.693±0.161*
Phosphorylated derivative 50 0.044±0.004*** 0.594±0.196
100 0.047±0.012** 0.674±0.099**
200 0.056±0.020** 0.804±0.109***
S180-bearing mice were administered p.o. with FT207 120 μg g−1 and carrageenan oligosaccharides and their derivatives 50, 100, and 200 μg g−1 for
14 days once daily. Normal control and S180-bearing control group received the same volume of distilled water. Values are mean±SD of six mice
n.d. not determined
*Significantly different from S180 control group at P<0.05
**Significantly different from S180 control group at P<0.01
***Significantly different from S180 control group at P<0.001
J Appl Phycol (2011) 23:59–65 63
Discussion
Clinical verification is being obtained, with a variety of
different therapeutic approaches, for the concept that
anticancer treatments based on exploiting the host’s own
antitumor defense mechanism can be beneficial (Ehrke
2003). The relationship between the occurrence, growth, and
decline of tumor and immune states is the essential problem
of tumor immunology and immunotherapy. The discovery
and identification of new antitumor drugs, which can
potentiate the immune function, has become an important
goal of research in immunopharmacology and oncotherapy.
Recently, a wide variety of antitumor activities of sulfated
oligosaccharides, such as antimetastasis, proliferation inhibi-
tion, and immune regulation, have been observed. In our
previous study, κ-carrageenan oligosaccharides were found
to have antitumor and immunomodulation effects on S180-
bearing mice. However, their antitumor activity was not as
effective as the chemotherapeutic drug FT207. So, we
hypothesized that chemical modification of carrageenan
oligosaccharides might enhance their antitumor activity. In
this study, we demonstrated that all the chemically modified
derivatives showed enhanced inhibitory effect compared to
carrageenan oligosaccharides, especially for the sulfated
derivative at a high dose. Furthermore, their immunostimu-
latory activity on macrophage phagocytosis and cellular
immunity, especially on spleen lymphocyte proliferation,
was also increased by chemical modification.
The exact mechanism of the positive effect of carra-
geenan oligosaccharide and their derivatives on immune
response in tumor-bearing hosts is not clear. As we know,
sulfated oligosaccharides can inhibit tumor metastasis and
promote tumor regression by their effects on heparanase
activity and angiogenic growth factor action. Oligosaccha-
ride chain length and the degree of sulfation are more
important parameters than the sugar composition and type
of linkage. With increasing sulfation, there is a steady
increase in the ability of maltohexaose to inhibit both
heparanase activity and fibroblast growth factor (FGF)
binding to heparin sulfate (Parish et al. 1999). In addition,
the oversulfated fucoidans showed more potential suppres-
sive effect on vascular endothelial growth factor (VEGF)165
than natural fucoidans, suggesting an important role for the
numbers of sulfate groups in the inhibitory role (Soeda et
al. 2000; Koyanagi et al. 2003). Carrageenans also have
been found to inhibit binding of basic fibroblast growth
factor (bFGF), transforming growth factor β1 (TGF β1),
and platelet-derived growth factor (PDGF; Hoffman 1993).
Carrageenans and modified carrageenans have been tested
for their ability to inhibit the formation of new blood
capillaries with good success; however, they had consider-
able side effects. Better results were obtained with a series
of modified galactans. Hence, β-1,4-galactansulfates of
relatively low degree of polymerization with a distinct
degree of sulfationhave better antiangiogenic activity,
which entitles them for further development as angiogen-
esis inhibitor lead structures (Franz et al. 2000). Chen et al.
(2007) found that the low sulfate content κ-carrageenan
oligosaccharides expressed no effect on angiogenesis, while
higher sulfate content λ-carrageenan showed the highest
antiangiogenic activity, which indicated that the degree of
sulfation is a critical structural parameter for the ability of
carrageenan oligosaccharides to inhibit angiogenesis.
Besides the degree of sulfation, the position of the sulfate
group is essential for the inhibition of angiogenesis. It
seems likely that a sulfate group at position 4 of the
galactose is important (Paper et al. 1995). It was also
found that the acetyl-iota-carrageenan and methyl-iota-
Group Dose (μg g−1 day−1) IL-2 (pg mL−1) TNF-α (pg mL−1)
Normal control – 92.8±17.7** 58.9±11.1**
S180 control – 56.9±9.5 32.8±9.4
FT207 120 51.2±10.3 30.5±7.0
Carrageenan oligosaccharides 50 74.8±5.5** 40.7±8.9
100 84.8±11.0** 42.3±11.6
200 87.9±15.9** 58.2±11.4**
Sulfated derivative 50 78.3±13.3* 25.3±9.9
100 72.7±10.8* 42.2±9.3
200 73.5±7.3* 59.0±14.8*
Acetylated derivative 50 69.7±10.3 22.7±10.8
100 74.0±15.2 32.5±9.6
200 89.7±17.2** 34.6±9.2
Phosphorylated derivative 50 60.6±9.1 25.3±10.4
100 79.9±18.1* 44.3±13.0
200 56.2±8.0 33.9±6.5
Table 4 Effects of carrageenan
oligosaccharides and their deriv-
atives on cytokine production in
serum of S180-bearing mice
S180-bearing mice were admin-
istered p.o. with FT207
120 μg g−1 and carrageenan
oligosaccharides and their deriva-
tives 50, 100, and 200 μg/g g−1
for 14 days once daily. Normal
control and S180-bearing control
group received the same volume
of distilled water. Values are
mean±SD of six mice
*Significantly different from
S180 control group at P<0.05
**Significantly different from
S180 control group at P<0.01
64 J Appl Phycol (2011) 23:59–65
carrageenan had the similar antiangiogenic effect as iota-
carrageenan (Paper et al. 1995). In the present study, we
also found the sulfated derivative showed significantly
higher antitumor and immunostimulatory activity, and that
the acetylated and phosphorylated derivatives had no
obvious effect compared with oligosaccharides.
In addition, there has been a rapid increase in understand-
ing the important role of the innate immune system in
recognition and therapy of cancer. NK cells are innate immune
effectors that serve a number of important roles in antitumor
immunity. NK cells were first described functionally by virtue
of their unique ability to rapidly lyse tumor cells without
previous sensitization and without restriction by the major
histocompatibility complex. Depletion of NK cells from mice
often reduces the resistance of the mice to transplanted tumor
cell lines. It was found that the oligosaccharides can enhance
the NK cell activity, both in cancer patients and animals. NK
cell activation was increased by low molecular weight
heparins in cancer patients and in an animal model of lung
cancer (Bobek et al. 2005). An oligosaccharide AbPS isolated
from the Chinese herbal medicine Achyranthes bidentata
with a molecular weight of only 1,360 Da induced a
significant enhancement of natural killer cell activity in
S180-bearing mice (Li 2000). In this study, sulfated
derivatives significantly augmented the NK cell activities
compared to the carrageenan oligosaccharides and even
exceeded that of the normal mice at high dose, which may be
the key role to the antitumor activity of them.
It is worth mentioning that the molecular weight of the
sulfated derivatives had been changed from 1.2 to 0.8 kDa
due to the sulfation reaction, while that of the acetylated
and phosphorylated derivatives had little or no change. It is
known that the molecular weight also has a great effect on
the relevant biological activity of sulfated oligosaccharides
besides the degree of sulfation and the sulfation position.
The antitumor and immunostimulatory activity of the
sulfated derivative may also relate to the decrease of its
molecular weight. The relationship between structure and
biological activity remain to be elucidated, and the exact
correlation between molecular weight and antitumor activ-
ity needs further investigation.
Acknowledgements This work was financially supported by the
Innovative Key Project of the Chinese Academy of Sciences (KZCX2-
YW-209), the National Project for public welfare marine affairs
(200705010), the Science and Technology Development Program of
Shandong Province (2008BS06005) and the Talent program of
Chinese Academy of Science (Dongbei Zhi Chun).
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J Appl Phycol (2011) 23:59–65 65
	Enhanced...
	Abstract
	Introduction
	Materials and methods
	Macrophage phagocytosis assay (carbon clearance)
	Lymphocyte proliferation assay
	Quantitative hemolysis of sheep red blood cells (QHS) assay
	Assay of natural killer (NK) cells activity
	Cytokine assay
	Statistical analysis
	Results
	Effects of carrageenan oligosaccharides derivatives on thymus, spleen indexes, and tumor weights
	Effects of carrageenan oligosaccharides derivatives on macrophage phagocytosis and humoral immunity
	Effects of carrageenan oligosaccharides derivatives on cellular immunity
	Effects of carrageenan oligosaccharides derivatives on cytokine production in serum
	Discussion
	References