Biological activities of fucose-containing polysaccharide ascophyllan isolated from the brown alga Ascophyllum nodosum

Article abstract of DOI:10.1271/bbb.80845

A fucose-containing, sulfated polysaccharide ascophyllan was isolated from the brown alga Ascophyllum nodosum. Composition analysis demonstrated that ascophyllan mainly contains uronic acid, xylose, fucose, and sulfate half ester in approximately equimole

Full text of DOI:10.1271/bbb.80845

Biosci. Biotechnol. Biochem., 73 (4), 961–964, 2009
Note
Biological Activities of Fucose-Containing Polysaccharide Ascophyllan Isolated
from the Brown Alga Ascophyllum nodosum
1
2
2
2;y
Seiichirou NAKAYASU, Ryo SOEGIMA, Kenichi YAMAGUCHI, and Tatsuya ODA
1
Research and Development Division, Hayashikane Sangyo Co., Ltd., Shimonoseki, Yamaguti 750-8608, Japan
Division of Biochemistry, Faculty of Fisheries, Nagasaki University, Nagasaki 852-8521, Japan
2
Received December 2, 2008; Accepted December 24, 2008; Online Publication, April 7, 2009
[doi:10.1271/bbb.80845]
A fucose-containing, sulfated polysaccharide asco-
acid (mannuronic acid) with fucose containing branches
7
–9)
phyllan was isolated from the brown alga Ascophyllum
nodosum. Composition analysis demonstrated that as-
cophyllan mainly contains uronic acid, xylose, fucose,
and sulfate half ester in approximately equimolecular
proportions, which are evidently distinct from those of
alginate and fucoidan. Ascophyllan inhibited the pro-
liferation of U937 cells in a concentration-dependent
manner, and DNA-fragmentation and typical apoptotic
nuclear morphological changes were observed in the
ascophyllan-treated cells. Furthermore, ascophyllan
induced the secretion of tumor necrosis factor-ꢀ
(3-O-D-xylosyl-L-fucose-4-sulfate).
In spite of in-
creasing study on the various biological activities of
fucoidans, there is no detailed study on the biological
activities of ascophyllan. Therefore, in this study, we
prepared ascophyllan from powdered A. nodosum based
7
–9)
on a method reported previously,
and explored its
biological activities on two cultured cell lines. To our
knowledge, this is the first time that the biological
activities of ascophyllan have been reported.
Fucoidan (from Fucus vesiculosus) was purchased
from Sigma (St. Louis, MO). Ascophyllum nodosum
collected on the coast of Norway was obtained from
KAISEI (Shimonoseki, Japan). For the preparation of
ascophyllan, fucoidan, and alginate fractions from
A. nodosum, 40 g of milled A. nodosum was pretreated
with diluted HCl (final 0.2 N) to remove inorganic
cations. After filtration, the residue was put into
1,000 ml of distilled water, then neutralized with
aqueous NaOH. This solution was gently stirred at
(
(
TNF-ꢀ) and granulocyte colony-stimulating factor
G-CSF) from mouse macrophage cell line RAW264.7.
Key words: Ascophyllum nodosum; ascophyllan; fucoi-
dan; apoptosis; cytokine
Various useful polysaccharides are available from
several species of marine algae, and it is well known that
some polysaccharides have a widevariety of biological
activities. Laminaran and especially fucoidan are draw-
ing increasing attention from medical and food supple-
ment areas for their potential biological activities.
Although the composition of these polysaccharides,
molecular sizes, and entire structures vary depending on
the algal species, the extraction procedure, and even the
ꢀ
20 C for 20 h, then filtrated. After the residue was again
put into 1,000 ml of distilled water, this solution was
ꢀ
stirred at 100 C for 2 h, then filtrated. The filtrates were
combined, and the solution was adjusted to pH 1.3 with
HCl. The precipitated alginate fraction was removed by
centrifugation, and the supernatant was subsequently
filtrated. The precipitate was dissolved in water, dia-
lyzed, and lyophilized to give the alginate fraction. The
filtrate was neutralized with NaOH. The neutralized
solution was concentrated by evaporation under reduced
pressure, then dialyzed extensively against distilled
water to remove generated salt by neutralization.
Subsequently, alginate lyase was added to the solution
1
)
season of harvest and local climate conditions, the
polysaccharides have a common structural feature.
Fucoidan is basically a sulphated fucan containing
fucose as a main component and sometimes containing
uronic acids, galactose, and xylose as minor composi-
2)
tions. Laminaran is composed of (1,3)-ꢀ-glucan with
)
ꢀ
ꢀ
(1,6) branching,3 and alginate is composed of man-
to degrade residual alginate. After incubation at 30 C
4
)
nuronic and guluronic acid with ꢀ(1,4)-linkages. In
addition to the well-known anticoagulant and antith-
rombotic activity, fucoidans act on the inflammation
and immune systems and have antitumor and antiviral
activities and antiproliferative and antiadhesive effects
on cells, and can interfere with the mechanisms involved
in fertilization. These activities and the structural aspects
of fucoidans have been reviewed in recent papers.
Ascophyllan is a fucan, and has similar but distinct
structure from fucoidans based on a backbone of uronic
for 20 h, the solution was dialyzed extensively against
distilled water. Five parts of this solution were then
mixed with 1 part of 1 M NaCl solution and 6 parts of
ethanol. This process of dissolution of the macromole-
cules in water and their precipitation with ethanol was
repeated twice. The precipitated ascophyllan fraction
was collected by centrifugation and the pellet was
dissolved in water, dialyzed extensively against water,
and lyophilized. After precipitation of the ascophyllan
fraction, the supernatant was concentrated, dialyzed
5
,6)
y
To whom correspondence should be addressed. Tel: +81-95-819-2831; E-mail: t-oda@nagasaki-u.ac.jp
Abbreviations: Z-D-CH2-DCB, carbobenzoxy-Asp-1-yl-[(2,6-dichlorobenzoyl)oxy] methane; TNF-ꢁ, tumor necrosis factor-ꢁ; G-CSF, granulocyte
colony-stimulating factor; ABEE, p-aminobenzoic ethyl ester; TFA, trifluoroacetic acid; WST-8, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-
(2,4-disulfophenyl)-2H-tetrazolium monosodium salt; FBS, fetal bovine serum; DMEM, Dulbecco’s modified Eagle’s minimal essential medium;
ELISA, enzyme-linked immunosorbent assay; Fuc, L-fucose; Xyl, D-xylose; Glu, D-glucose; Man, D-mannose; Gal, D-galactose; UA, uronic acid;
2
ꢁ
SO4 , ester sulfate group

962
S. NAKAYASU et al.
Table 1. Composition (%) of Ascophyllan, Fucoidan, Alginate Fractions, and Commercially Available Fucoidan (Sigma) and Molar Ratios of the
Compositions
Composition of neutral sugarsa
UAb
2ꢁ
SO4
c
Polysaccharide
Fuc
Xyl
Glu
Man
Gal
Ascophyllan
Fucoidan
Alginate
15.5 (1)
28.4 (1)
0.5 (1)
13.4 (0.95)
4.3 (0.16)
0.4 (0.79)
1.9 (0.09)
0.3 (0.02)
2.0 (0.06)
0.1 (0.16)
0.8 (0.03)
3.4 (0.2)
0.6 (0.04)
5.3 (0.17)
1.6 (2.99)
3.1 (0.11)
21.4 (1.17)
5.8 (0.17)
27.8 (47.4)
9.6 (0.33)
9.6 (1.06)
19.4 (1.17)
0 (0)
0.8 (0.03)
0.6 (1.07)
1.0 (0.04)
d
Sigma fucoidan
24.8 (1)
22.6 (1.56)
aDetermined by HPLC assay after acid hydrolysis.
bDetermined by carbazole method and calculated as glucuronic acid equivalent.
cDetermined by turbidimetric assay after acid hydrolysis.
dPurified from Fucus vesiculosus.
The numbers in the brackets indicate the molar ratios of the compositions of the polysaccharides, calculated by taking fucose content as 1.
A
B
M
(a) (b)
1
00
(
bp)
8
6
4
2
0
0
0
0
0
4
2
,870
,016
1
,360
1
,107
9
6
26
58
489
2
67
62.5
125
250
500
1000
Concentration (µg/ml)
C
D
3
2
2
1
1
0
5
0
5
0
5
0
10 µm
Control Ascophyllan
Fig. 1. Cytotoxic Effects of Three Polysaccharide Fractions Isolated from A. nodosum on U937 Cells.
5
A, U937 cells in 24-well plates (3 ꢂ 10 cells/well) were incubated with the indicated concentration of alginate ( ), fucoidan ( ), and
ꢀ
ascophyllan ( ) fractions in RPMI1640 medium containing 10% FBS at 37 C for 3 d. The viabilities of the treated cells were examined by
WST-8 assay, as described in the text. Columns represent average of triplicate measurements and bars indicate the standard deviation. B, U937
5
cells in 24-well plates (3 ꢂ 10 cells/well) were cultured without (a) and with (b) 1,000 mg/ml of ascophyllan in RPMI1640 medium containing
ꢀ
1
0% FBS at 37 C for 3 d. DNA was extracted from treated cells and analyzed by electrophoresis in 2.0% agarose gel, as described in the text.
6
C, U937 cells (1 ꢂ 10 cells/ml) in 35-mm diameter dish in RPMI1640 medium containing 10% FBS were cultured with and without
ꢀ
ascophyllan (final 1,000 mg/ml) for 3 d at 37 C in the presence ( ) and the absence ( ) of caspase family protease inhibitor, Z-D-CH2-DCB
(
final 100 mM), and then diphenylamine assay was performed as described in the text. Columns represent average of triplicate measurements and
4
bars indicate standard deviations. D, Cells (2 ꢂ 10 cells/well) in 96-well plates in RPMI1640 medium containing 10% FBS were cultured with
ꢀ
ꢀ
ascophyllan (final 1,000 mg/ml) for 3 d at 37 C. Hoechst 33258 (final 1 mM) was added to the treated cells and incubated for 5 min at 37 C, and
then fluorescence microscopic observation was conducted using a fluorescent microscope. Arrow indicates a cell with typical nuclear apoptotic
change.
extensively against water, and lyophilized to give the
fucoidan fraction. For sugar composition analysis, the
p-aminobenzoic ethyl ester (ABEE)-labeling procedure
was employed, as previously reported. Uronic acid
was determined by a modified carbazole-sulfuric acid
method, and was calculated as glucuronic acid.11) Ester
sulfate was determined by the method of Dodgson-
Price’s turbidimetry.
A human myeloid leukemia U937 cell line was
obtained from the Riken Cell Bank (Tsukuba, Japan).
1
2)
1
0)

Biological Activities of Ascophyllan
963
ꢀ
The cells were cultured at 37 C in RPMI1640 medium
Gibco Grand Isle, NY) supplemented with 10% fetal
bovine serum (FBS) in a humidified atmosphere of 5%
A
(
5
4
3
2
1
0
CO and 95% air. RAW264.7 (mouse macrophage) cells
2
were obtained from the American Type Culture Collec-
tion (Rockville, MD), and cultured in Dulbecco’s
modified Eagle’s minimum essential medium (DMEM)
supplemented with 10% fetal bovine serum (FBS),
penicillin G sodium (100 units/ml), and streptomycin
sulfate (100 mg/ml). The cytotoxicity of each polysac-
charide fraction with various concentrations (0–1,000
mg/ml) on U937 cells was measured by 2-(2-methoxy-4-
nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-
tetrazolium monosodium salt (WST-8) assay. Ascophyl-
lan-induced DNA fragmentation in U937 cells was
0
62.5
125
250
500
1000
Ascophyllan (µg/ml)
B
25
1
3,14)
examined as described previously.
Nuclear mor-
20
phological changes were examined by staining with the
DNA-binding fluorochrom bisbenzimide (Hoechst
15
3
3258) as previously described.14) The levels of TNF-ꢁ
1
0
5
0
and G-CSF in the supernatants of RAW264.7 cells
cultured with ascophyllan were measured by sandwich
enzyme-linked immunosorbent assay (ELISA) with
two antibodies to two different epitopes on each
cytokine molecule by similar methods, as described
0
62.5
125
250
500
1000
previously.15)
Ascophyllan (µg/ml)
By taking advantage of the solubility of fucans in
acidic medium and use of alginate lyase, alginates were
removed from the fucan fractions. Subsequently, using
the different solubility between ascophyllan and fucoi-
dan in aqueous ethanol, ascophyllan was separated from
the fucoidan fraction. The yields of alginate, fucoidan,
and ascophyllan fractions obtained from 40 g of milled
A. nodosum were 4.7 g, 0.50 g, and 0.76 g, respectively.
Composition analysis of the three polysaccharide frac-
tions showed that each of them had a characteristic
composition feature (Table 1). Ascophyllan had fucose
and xylose in about equimolecular proportion, whereas
fucoidan had much higher ratio of fucose than of xylose,
and the contents of these monosaccharides in the
alginate were very low. The monosaccharide composi-
tion profile of fucoidan fraction obtained in this study
was similar to that of a commercially available fucoidan.
Significant levels of sulphate half-ester groups were
detected in the ascophyllan and fucoidan fractions, but
no level was detectable in the alginate fraction. The
level of uronic acid in ascophyllan fraction was much
higher than that of fucoidan. Based on the molar ratios
calculated by taking fucose content as 1 (Table 1),
ascophyllan is suggested to be a heteropolysaccharide
Fig. 2. Ascophyllan-Induced Cytokine Secretion from RAW264.7
Cells.
4
Adherent cells (2 ꢂ 10 cells/well) in 96-well plates were
incubated with the indicated concentrations of ascophyllan in
ꢀ
DMEM containing 10% FBS at 37 C. After 24 h, the cytokine
levels in the supernatant were measured by the sandwich ELISA
method, as described in the text. Columns represent averages of
triplicate measurements and bars indicate the standard deviations.
duction of apoptosis. To our knowledge, this is the first
report suggesting that ascophyllan induces apoptosis in
U937 cells. Since ascophyllan-iduced DNA fragmenta-
tion was strongly inhibited by the caspase family
inhibitor, perhaps the caspase-activation pathway was
involved. Recent study has demonstrated that a com-
mercially available fucoidan (Sigma, St. Louis, MO)
purified from Fucus vesiculosus induced apoptosis in
human myeloma and leukemia cells through activation
of the caspase-3 and down-regulation of ERK path-
1
6)
ways. In addition, Riou et al. reported that fucoidan
extract obtained from Ascophyllum nodosum directly
inhibited the proliferation of a human non-small cell
1
7)
lung cancer cell line (NSLC-N6). Thus, it seems likely
that sulfated fucans like fucoidans and ascophyllan are
able to cause direct cytotoxic effects on target cells,
including tumor cells, through induction of apoptosis.
Regarding antitumor activity in in vivo systems, it has
been reported that fucoidan fractions extracted from
Sargassum thunbergii and Sargassum kjellmanianum
showed antitumor effects for Eherlich carcinoma-
bearing mice and L1210 leukemia-bearing mice, re-
(
xylofucoglycuronan) consisting of fucose, xylose,
uronic acid, and sulphate half-ester in approximately
equimolecular proportion, consistently with previous
reports,7 and the composition was obviously distinct
from those of fucoidan and alginate fractions.
,9)
To examine the cytotoxic effects of the fucoidan,
ascophyllan, and alginate fractions on U937 cells,
WST-8 assay was carried out. As shown in Fig. 1A,
fucoidan and ascophyllan showed similar cytotoxic
effects on U937 cells, while alginate had no toxic effect.
The cytotoxicity of ascophyllan was concentration-
dependent, and even at 62.5 mg/ml, more than 50% of
cells died after 72 h incubation. DNA-fragmentation
assay and fluorescent nuclear staining suggested that the
cytotoxicity of ascophyllan was accompanied by in-
1
8,19)
spectively.
In these in vivo experimental models,
perhaps fucoidan-induced enhancement of immune
responses is involved in the anti-cancer effects of
fucoidan. Previous studies have pointed out that fucoi-
dans or sulfated fucans can act as immunological
1
7,20)
response modifiers.
To gain insight into this aspect
of the biological activity of ascophyllan, we investigated
the effects of ascophyllan on mouse macrophage cell

9
64
S. NAKAYASU et al.
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ascophyllan induced the secretion of TNF-ꢁ and G-
CSF from RAW264.7 cells in a concentration-dependent
manner (Fig. 2). Similarly to ascophyllan, it has been
shown that fucoidan can stimulate TNF-ꢁ release from
monocytes.2 Therefore, ascophyllan may have various
biological activities similar to fucoidan, and it is
possible that ascophyllan can exert anti-tumor activity
through activation of host immune systems in addition
to the direct cytotoxic effects on target cells. To clarify
this point, further studies are necessary to investigate the
anti-tumor effects of ascophyllan using tumor-bearing
mice models.
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