Structure and anticancer activity of native and modified polysaccharides from brown alga Dictyota dichotoma

Article abstract of DOI:10.1016/j.carbpol.2017.10.006

The laminaran DdL and fucoidan DdF were obtained from the brown alga Dictyota dichotoma. DdF was a sulfated (28.9%) and acetylated heteropolysaccharide containing fucose, galactose, mannose and glucose (57.9, 20.4, 12.4 and 9.2 mol%, respectively). DdL was a 1,3;1,6-β-D-glucan with the main chain built from 1,3-linked glucose residues and single glucose residue in branches at C6 (one branch on three glucose residues of the main chain). Sulfated (43.7%) laminaran DdLs was obtained from DdL by sulfation. It was determined that sulfates occur at C2, C4 and C6 of glucose residues. The anticancer effect of DdF, DdL, and DdLs (200 μg/mL) was studied in vitro on colon cancer cells HCT-116, HT-29, and DLD-1. The effect of polysaccharides (40 μg/mL) on colony formation of DLD-1 cancer cells after irradiation (4 Gy) was investigated first. All polysaccharides showed a synergistic effect with X-ray irradiation against cancer cells, decreasing the amount and size of cancer cells colonies.

Full text of DOI:10.1016/j.carbpol.2017.10.006

CarbohydratePolymers180(2018)21–28
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Research paper
Structure and anticancer activity of native and modified polysaccharides
from brown alga Dictyota dichotoma
Roza V. Usoltseva⁎, Natalia M. Shevchenko, Olesya S. Malyarenko, Irina A. Ishina,
Svetlana I. Ivannikova, Svetlana P. Ermakova
G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Laboratory of Enzyme Chemistry, 159 100-Let Vladivostoku
Ave., 690022, Vladivostok, Russian Federation
A R T I C L E I N F O
A B S T R A C T
Keywords:
Dictyota
Fucoidan
Laminaran
Structure
Anticancer activity
γ-radiation
The laminaran DdL and fucoidan DdF were obtained from the brown alga Dictyota dichotoma. DdF was a sulfated
(28.9%) and acetylated heteropolysaccharide containing fucose, galactose, mannose and glucose (57.9, 20.4,
12.4 and 9.2 mol%, respectively). DdL was a 1,3;1,6-β-D-glucan with the main chain built from 1,3-linked
glucose residues and single glucose residue in branches at C6 (one branch on three glucose residues of the main
chain). Sulfated (43.7%) laminaran DdLs was obtained from DdL by sulfation. It was determined that sulfates
occur at C2, C4 and C6 of glucose residues. The anticancer effect of DdF, DdL, and DdLs (200 μg/mL) was studied
in vitro on colon cancer cells HCT-116, HT-29, and DLD-1. The effect of polysaccharides (40 μg/mL) on colony
formation of DLD-1 cancer cells after irradiation (4 Gy) was investigated first. All polysaccharides showed a
synergistic effect with X-ray irradiation against cancer cells, decreasing the amount and size of cancer cells
colonies.
1. Introduction
Aziz, & Salem, 1980; Men’shova et al., 2012), P. tetrastromatica
(Karmakar et al., 2009; Rao, Sastry, & Rao, 1984), Spatoglossum
Fucoidans are the sulfated algal polysaccharides, which exhibit a
wide spectrum of biological activitity, including immunomodulatory,
anticoagulant, anticancer, radioprotective and antiviral properties (Ale,
Mikkelsen, & Meyer, 2011; Kusaykin et al., 2008). Additionally, fucoi-
dans are non-toxic to organisms and have no side effects. Substances
possessing these properties can be used in combined cancer therapy,
which is comprised of surgical, radiation and medicinal treatment.
There are only a few studies devoted to the radioprotective activity of
fucoidans (Byon et al., 2008; Lee et al., 2008; Lee, Bae, Cho, & Rhee,
2009; Qiong et al., 2011), although these polysaccharides are surely
prospective non-toxic radioprotectors.
schroederi (Leite et al., 1998; Rocha et al., 2005), Stoechospermum
marginatum (Adhikari et al., 2006) – produce predominantly hetero-
geneous fucoidans. Polysaccharides from D. dichotoma have been ob-
tained and investigated by some research groups (Abdel-Fattah,
Hussein, & Fouad, 1978; Hussein, Fouad, & Abdel-Fattah, 1979;
Rabanal, Ponce, Navarro, Gomez, & Stortz, 2014). It was shown that
obtained fucoidans are sulfated heteropolysaccharides. The system of
fucoidans (more than 60 fractions), containing fucose, galactose,
mannose, xylose, glucose, rhamnose, arabinose and uronic acid re-
sidues, was obtained and investigated in study (Rabanal, Ponce,
Navarro, Gomez, & Stortz, 2014).
The brown algae Dictyota dichotoma belongs to the family
Although fucoidans from this alga have been characterized, there
are no data on the isolation and structural characterisation of other
algal polysaccharides – laminarans in the literature sources. The lami-
narans (1,3;1,6-β-D-glucans) are also of interest due to their biological
effects and non-toxicity. Earlier, we established that chemical and en-
zymatic modification of laminarans can improve the biological activity
of derivatives in comparison with native polysaccharides (Elyakova
et al., 2007; Malyarenko et al., 2017; Menshova et al., 2014;
Zvyagintseva, Elyakova, & Isakov, 1995;).
Dictyotaceae (order Dictyotales). The algae from this order
–
Canistrocarpus cervicornis (Camara et al., 2011), Dictyopteris delicatula
(Magalhaes et al., 2011), D. plagiogramma (Percival, Rahman, & Weigel,
1981),
D.
polypodioides
(Sokolova,
Ermakova,
Awada,
Zvyagintseva, & Kanaan, 2011), Dictyota dichotoma (Abdel-Fattah,
Hussein, & Fouad, 1978; Hussein, Fouad, & Abdel-Fattah, 1979;
Rabanal, Ponce, Navarro, Gomez, & Stortz, 2014), D. menstrualis
(Albuquerque et al., 2004), Lobophora variegata (Medeiros et al., 2008),
Padina gymnospora (Silva et al., 2005), P. pavonica (Hussein, Abdel-
Thus, the aim of this work was to investigate the structural
⁎
Corresponding author.
E-mail address: Usoltseva-R@yandex.ru (R.V. Usoltseva).
http://dx.doi.org/10.1016/j.carbpol.2017.10.006
Received 23 June 2017; Received in revised form 4 August 2017; Accepted 2 October 2017
Availableonline04October2017
0144-8617/©2017ElsevierLtd.Allrightsreserved.

R.V. Usoltseva et al.
CarbohydratePolymers180(2018)21–28
characteristics of polysaccharides from Dictyota dichotoma, to modify
the native laminaran, and to study the anticancer and radioprotective
activities of fucoidan, laminaran and its sulfated derivative.
dextrans of molecular weights 5, 6, 10, 40, 70, 100, 200 and
450–650 kDa as reference standards.
2.3.3. Polysaccharide extraction
2. Experimental
Samples of defatted, dried and powdered algal fronds (100 g) were
extracted twice with 0.1 M HCl (2 L) for 2 h at 60 °C. The extracts were
combined, centrifuged, dialyzed, concentrated on a rotary evaporator
(2.5 h, 45 °C) and lyophilized to obtain the polysaccharide fraction DdP
(8.4 g).
2.1. Materials
Organic solvents, inorganic acids and salts, sodium hydroxide and
trifluoroacetic acid (TFA) were commercial products (Laverna-Lab,
Moscow, Russia). Standards (mannose, rhamnose, glucose, galactose,
xylose and dextrans) were purchased from Sigma–Aldrich (St. Louis,
MO, USA). Sorbents for chromatography were Polychrome-1 (Reakhim,
Moscow, Russia), DEAE-cellulose Sigma–Aldrich (St. Louis, MO, USA),
Macro-Prep DEAE (Bio-Rad Laboratories, Hercules, CA, USA) and
Amberlite CG-120 (Serva Electrophoresis GmbH, Heidelberg,
Germany).
2.3.4. Anion-exchange chromatography of polysaccharides on DEAE-
cellulose
A solution of polysaccharide DdP in 50 mL of 0.1 M HCl was applied
to a DEAE-cellulose column (Cl– form, 21 × 3 cm) equilibrated with
0.1 M HCl. The laminaran-containing fraction was eluted with water,
neutralized and concentrated on rotary evaporator (40 min, 45 °C).
Then, the column was successively washed with 1 and 2 M NaCl solu-
tions until the disappearance in the eluate of a positive reaction for
carbohydrates by the phenol-sulfuric acid method (Dubois et al., 1956)
in each case. The fraction containing fucoidan was eluted with 2 M
NaCl, then concentrated on rotary an evaporator (40 min, 45 °C), dia-
lyzed and lyophilized with a yield of 1.1 g.
Basal Medium Eagle (BME), McCoy's 5A Modified Medium (McCoy's
5A), RPMI-1640 Medium, trypsin, fetal bovine serum (FBS) and agar
were purchased from Gibco/Life Technologies (Carlsbad, CA, USA).
MTS reagent
– 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide was purchased from Promega Corporation (Madison, WI,
USA). Phosphate buffered saline (PBS), L-glutamine and penicillin–-
streptomycin solution (10000 U/mL, 10 μg/mL) were from
Sigma–Aldrich (St. Louis, MO, USA).
2.3.5. Hydrophobic chromatography of laminaran on Polychrome-1
The solution of laminaran was applied to a Polychrome-1 column
(15 × 4.5 cm). The column was subsequently washed with water and
5% aqueous ethanol. Then, the pure laminaran (L) DdL was eluted with
15% aqueous ethanol until the disappearance in the eluent of a positive
reaction for carbohydrates by the phenol-sulfuric acid method (Dubois
et al., 1956). The eluate was concentrated on a rotary evaporator
(40 min, 45 °C) and lyophilized. The yield of the laminaran fraction DdL
was 1.3 g.
Human colorectal adenocarcinoma HCT-116 (ATCC® no. CCL-
247™), HT-29 (ATCC® no. HTB-38™), DLD-1 (ATCC® no. CCL-221™) cell
lines were obtained from the American Type Culture Collection
(Manassas, VA, USA).
A sample of the algae Dictyota dichotoma (Dd) was collected from
Peter the Great Bay in August 2014, Sea of Japan (Russia). Fresh algal
biomass (100 g) was powdered and pre-treated with 70% aqueous
ethanol (w/v = 1:10) at room temperature for 10 days. Defatted alga
was air-dried.
2.2. Instruments
2.3.6. Removal of polyphenols from the fucoidan fraction
A sample of the fucoidan fraction was dissolved in water (100 mL),
to which was added 30% aqueous H2O2 (20 mL), and then 10% aqueous
NH3 until to the solution reached pH 8.5. The resulting mixture was
kept at room temperature for 17 h in the dark. Then, the solution was
centrifuged, and the supernatant was dialyzed, concentrated on a rotary
evaporator (40 min, 45 °C) and lyophilized to obtain the pure fucoidan
fraction DdF with a yield of 739 mg.
NMR spectra were obtained on an Avance DPX-500 NMR spectro-
meter (Bruker BioSpin Corporation, Billerica, MA, USA) resonating at
75.5 MHz at 35 and 60 °C. The sample concentration was 15 mg of
polysaccharide/mL of D2O for 1D and 2D experiments.
2.3. General methods
2.3.1. Analytical procedures
2.3.7. Anion-exchange chromatography of fucoidan on Macro-Prep DEAE
A solution of fucoidan in 0.1 M NaCl (0.5 g in 10 mL) was applied to
a Macro-Prep DEAE (Bio-Rad, USA) column (Cl– form, 2.5 × 9 cm)
equilibrated with 0.1 M NaCl. Then the column was successively eluted
with a linear gradient of NaCl (from 0.1 to 2 M). The eluate was ana-
lyzed by the phenol-sulfuric acid method (Dubois et al., 1956). The
obtained fraction of fucoidan DdF was concentrated on a rotary eva-
porator (40 min, 45 °C), dialyzed and lyophilized with a yield of
415 mg.
Total carbohydrates were quantified by the phenol-sulfuric acid
method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). Mono-
saccharide composition was determined by HPLC with an ISA-07/
S2504 column (0.4 × 25 cm, Shimadzu, Kyoto, Japan), a bicinchoni-
nate assay and a C-R2 AX integrating system (Shimadzu, Kyoto, Japan)
after hydrolysis with 2 M trifluoroacetic acid (6 h, 100 °C). Mono-
saccharides (rhamnose, ribose, mannose, fucose, galactose, xylose, and
glucose) were used as standards for HPLC. The protein and polyphenol
contents were determined using the Bradford method (Bradford, 1976)
and a modification of the Folin–Ciocalteau method (Singleton & Rossi,
1965), respectively. The amount of sulfate groups was determined by
using the BaCl2 gelatin method (Dodgson, 1961).
2.3.8. Sulfation of laminaran
Pyridine (30 mL) in a bottle was cooled in an ice bath, and chlor-
osulfonic acid (10 mL) was slowly dropped into the pyridine solution
and incubated for 1 h to form a mixture. The laminaran DdL (1 g) was
dissolved in 100 mL of dimethylformamide, and the resulting solution
was added to the mixture. Then, the bottle with reagents was kept in a
hot water bath at 75 °C for 1.5 h. Subsequently, the solution was cooled,
resolved in water (250 mL) and neutralized with NaOH. The obtained
sulfated (s) laminaran DdLs was precipitated by 96% ethanol and
centrifuged. The precipitate was dissolved in water, dialyzed and lyo-
philized with a yield of 377 mg.
2.3.2. Molecular weight determination
Samples of polysaccharides were analyzed using a high-perfor-
mance liquid chromatography instrument (Agilent 1100 Series,
Germany) equipped with a refractive index detector and a series-con-
nected gel-filtration column (TSK gel G4000 SW and TSK gel G2000
SW, Tosoh Co., Tokyo, Japan). Elution was performed with a 0.05 M
aqueous solution of Na2SO4 at 50 °C with a flow rate of 0.5 mL/min.
The molecular weights of polysaccharides were estimated using
22

R.V. Usoltseva et al.
CarbohydratePolymers180(2018)21–28
2.4. Biological assays
subsequently purified by anion-exchange chromatography on Macro-
Prep DEAE. The obtained fucoidan fraction DdF was 0.4% of defatted
algae weight.
2.4.1. Cell culture
Human colorectal adenocarcinoma cells HCT-116 and HT-29 were
cultured in McCoy's 5A medium; DLD-1 cells were maintained in RPMI-
1640 medium. Culture media were supplemented with 10% FBS and
penicillin–streptomycin solution. The cell cultures were maintained at
37 °C in a humidified atmosphere containing 5% CO2.
Analysis of the monosaccharide composition of polysaccharide
fractions revealed that DdL is pure glucan, and DdF is a hetero-
polysaccharide containing fucose, galactose, mannose and glucose
(57.9, 20.4, 12.4 and 9.2 mol%, respectively). Based on HPLC, the
molecular weight interval was 5–6 kDa for laminaran and 250–280 kDa
for fucoidan fractions.
2.4.2. Soft agar assay
The sulfate content of DdF was 28.9% of the total sample weight.
Earlier fucoidans from the brown algae D. dichotoma were investigated
by other groups (Abdel-Fattah et al., 1978; Hussein et al., 1979;
Rabanal et al., 2014). It was shown that this alga produce sulfated
heteropolysaccharides, containing fucose, galactose, mannose, xylose
and glucuronic acid. Our results are in good agreement with published
data.
Cells (8.0 × 103) were seeded in 6-well plate and treated with the
polysaccharides (laminarans, their sulfated derivative and fucoidans)
(40 or 200 μg/mL) in 1 mL of 0.3% BME agar containing 10% FBS,
2 mM L-glutamine and 25 μg/mL gentamicin. The cultures were
maintained at 37 °C in a 5% CO2 incubator for 14 days, and the cell
colonies were scored using a microscope Motic AE 20 (Xiamen, China)
and the Motic Image Plus computer program.
The fucoidan DdF has a very complex and difficult to elucidate 13C
NMR spectrum. This spectrum contains intensive signals at 16.2–16.5
and 61–62 ppm corresponding to C6 of α-L-fucose and unsulfated ga-
lactose, mannose or glucose, respectively. Signals corresponding to
acetyl groups and uronic acids are at 21–22 and 175–176 ppm.
2.4.3. Radiation exposure
Irradiation was deliver at room temperature using single doses from
the X-ray system XPERT 80 (KUB Technologies, Inc, Milford, CT, USA).
The applied doses were from 4 to 8 or 4 Gy for the colony formation
assay. The absorbed dose was measured using an X-ray radiation clin-
ical dosimeter DRK-1 (Axelbant LLK, Moscow, Russia).
3.2. The structure of native and sulfated laminaran
2.4.4. Cell irradiation
Laminarans are water-soluble polysaccharides of brown algae,
consisting of 1,3- and 1,6-linked b-D-glucose residues. Laminarans from
different species of algae are known to vary due to the ratio of 1,3:1,6
bonds and variants of including of these bonds in the molecule of β-D-
glucan (Zvyagintseva, Shirokova, & Elyakova, 1994). Laminarans
usually have a molecular weight of 4–5 kDa and contain a main chain
from 1,3-linked b-D-glucose residues with a small number (≤10%) of
branches at С6 as single b-D-glucose residues.
Cells were cultured in a sterile environment and kept in an in-
cubator at 5% CO2 and 37 °C to promote growth. Cell stocks were sub-
cultured every 3–4 days by rinsing the cells in phosphate buffered
saline (PBS), adding trypsin to detach the cells from the tissue culture
flask, and transferring 10–20% of the harvested cells to a new flask
containing fresh growth media.
Cells (5.0 × 105) were seeded in 60 mm dishes and exposed to ra-
diation at a dose rate from 4 to 8 Gy at 24 h after plating. Immediately
after irradiation, cells were returned to the incubator for recovery.
Three hour late cells were harvested and used for the soft agar assay to
establish the effect of radiation on colony formation of DLD-1 cells.
Cells (5.0 × 105) were plated in 60 mm dishes and incubated for
24 h. After the incubation, cells were exposed either in the presence or
absence of 40 μg/mL polysaccharides (native and sulfated laminarans
and fucoidan) for an additional 24 h before irradiation at a dose of
4 Gy. Immediately after irradiation, cells were returned to the incubator
for recovery. Three hours later, cells were harvested and used for the
soft agar assay to establish the effect of polysaccharides and radiation
on colony formation of DLD-1 cells.
The structure of laminaran DdL was investigated by 1D (1H, 13C)
and 2D (COSY, HSQC, HMBC) NMR spectroscopy. The 1H NMR spec-
trum of DdL contains group of two doublets at 4.76 and 4.78 ppm
(residues A and A’), and one doublet at 4.53 ppm (residue B) in
anomeric region. A full analysis of 2D spectra COSY and HSQC (Fig. 1A)
allowed to the determination of glucose proton signals and C/H cor-
relation. The chemical shifts of this laminaran are shown in Table 1.
The HSQC spectrum showed that glycosidic bonds are in position 3
of residue A and positions 3 and 6 of residue A’ (downfield location of
H3/C3 resonances of A and A’ at 3.79/86.0 and 3.79/85.5 ppm, re-
spectively, and H6/C6 resonances of A’ at 4.22, 3.88/69.9 ppm). The
analysis of HMBC spectrum (Fig. 1B) confirmed of these results. Cor-
relation peaks H1(A)/C3(A’) and H1(A’)/C3(A) at 4.78/85.5 and 4.76/
86.0 corresponded to the presence of 1,3-bonds between A and A’ re-
sidues. Cross-peak H1(B)/C6(A’) at 4.53/69.9 showed that glucose re-
sidue B was linked to C6 of residues A’.
3. Results and discussion
3.1. Purification and structural characteristics of polysaccharides from
brown alga D. dichotoma
The ratio of bonds 1,3:1,6 in the laminaran DdL was estimated by
comparing the intensity of anomeric proton signals in 1H NMR spec-
trum (group of two doublets belonged to residues A and A’ and the
doublet of residue B) and amounted to 3:1 (Kim et al., 2000).
Thus, our investigation showed that laminaran DdL is a regular
polysaccharide, which contain a main chain build from 1,3-linked
glucopyranose residues (A and A') with the branches of single glucose
(B) at C6 (ratio of bonds 1,3:1,6 = 3:1). The structure of DdL is pre-
sented in Fig. 2.
In this investigation, regular laminaran DdL with a determined
structure was sulfated (s) to obtain the laminaran derivative DdLs with
yield of 38% of native polysaccharide weight. The degree of sulfation of
sulfated laminaran was 43.7%. The structure of DdLs was investigated
by 13C NMR spectroscopy. The downfield location of signals C2, C4 and
C6 at 80–80.5, 75.8–76.2 and 68.8 ppm corresponded that DdLs has
sulfate groups at position 2, 4 and 6. The molecular weight range of the
sulfated laminaran fraction was 9–12 kDa.
Water-soluble polysaccharides were isolated from the brown alga
Dictyota dichotoma collected from Peter the Great Bay, Sea of Japan, Far
East of Russia. We used anion-exchange chromatography on DEAE-
cellulose for separation of the laminaran and fucoidan fractions. The
laminaran fraction was eluted by water, the fraction contained man-
nuronic acid and low-sulfated heterogeneous fucoidan – by 1 M NaCl,
and high-sulfated pure fucoidan – by 2 M NaCl. Then, the laminaran
was further purified by hydrophobic chromatography on Polychrome-1
to obtain the fraction DdL (1.3% of defatted algae weight).
Analysis of polyphenol content showed that the fucoidan fraction
eluted by 2 M NaCl from a DEAE-cellulose column contained a sig-
nificant amount of polyphenols (3.2% of sample weight). We removed
polyphenols from the fucoidan fraction according to known method
(Urvantseva et al., 2004). Earlier, this method was applied successfully
to fucoidan from the brown algae Fucus evanescens. Fucoidan was
23

R.V. Usoltseva et al.
CarbohydratePolymers180(2018)21–28
Fig. 1. HSQC (A) and HMBC (B) NMR spectra of
laminaran DdL.
Table 1
NMR data for the laminaran DdL.
Fragment of structure
С1
С2
С3
С4
С5
С6
A → 3)-β-D-Glcp-(1 → 3)-
B β-D-Glcp-(1 → 6)-
A’ → 3,6)-β-D-Glcp-(1 → 3)-
103.5
104.0
103.5
Н1
74.3
74.3
74.3
Н2
86.0
74.4
85.5
Н3
69.4
70.9
69.4
Н4
76.8
77.1
75.9
Н5
61.8
61.8
69.9
Н6
Fig. 2. Structure of the laminaran DdL.
A → 3)-β-D-Glcp-(1 → 3)-
B β-D-Glcp-(1 → 6)-
4.78
4.53
4.76
3.57
3.31
3.57
3.79
3.55
3.79
3.53
3.40
3.53
3.51
3.46
3.70
3.93; 3.74
3.93; 3.74
4.22; 3.88
A’ → 3,6)-β-D-Glcp-(1 → 3)-
The results revealed that all investigated polysaccharides showed
slight inhibition of colony formation of HCT-116 and HT-29 cells: less
than 10% compared with non-treated cells (control) for DdL and DdLs
for both cell lines; and 6% and 26% for HCT-116 and HT-29 cells, re-
spectively, for DdF. The highest inhibitory activity of these poly-
saccharides was observed for DLD-1 cells.
3.3. Biological activity of polysaccharides
We determined the effect of the polysaccharides DdL, DdLs and DdF
(at a concentration of 200 μg/mL) on colony formation of human colon
cancer cells HCT-116, HT-29, and DLD-1 using the soft agar clonogenic
assay (Fig. 3).
The percentage of inhibition was approximately 50% for DdF, and
sulfated laminaran DdLs also possessed a powerful inhibitory activity:
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R.V. Usoltseva et al.
CarbohydratePolymers180(2018)21–28
from S. cichorioides and its sulfated derivative suppressed colony for-
mation of colon cancer HCT-116 cells by 16 and 32%, respectively,
compared with non-treated cells (Malyarenko et al., 2017).
The fucoidans from brown algae differ widely in their structural
characteristics, and their anticancer activity can depend on their mo-
lecular weight, monosaccharide composition, branching and content of
sulfated and acetyl groups. The specific relationship between fucoidan
structure and anticancer effect are not determined. For example, ga-
lactofucans from Sargassum duplicatum, Sargassum feldmannii, Padina
boryana and Dictyota divaricata with different content of galactose,
sulfate and acetyl groups have widely variable anticancer activity in
vitro (Shevchenko et al., 2017). While structural simplification of fu-
coidan from Saccharina gurjanovae by of lowering its molecular weight,
desulfation at C2 and removal of galactose residues did not decrease its
anticancer activity (Shevchenko et al., 2015). Fucoidans from the
brown algae S. cichoriodes and D. dichotoma were very different in their
structure, and their biological effects were also different.
The published data about the effect of laminarans on colony for-
mation of cancer cells is very limited. It is known that laminaran from
brown seaweed Laminaria digitata (commercial preparation from
Sigma–Aldrich) was found to inhibit cell growth of colorectal cancer
cells HT-29 by 40% with lower cytotoxicity on IEC-6 intestinal epi-
thelial cells (Park, Kim, Kim, & Nam, 2012; Park, Kim, Kim, & Nam,
2013). Another group of authors showed that the same laminaran
suppressed proliferation of colorectal cancer cells LOVO by 38.8%.
Moreover, they obtained sulfated laminaran and determined its anti-
proliferative activity in LOVO cells. It was found that after sulfated
modification the growth inhibitory activity of sulfated laminaran was
enhanced, and the inhibition rate was 86% at the same dose (Ji et al.,
2012; Ji, Ji, & Meng, 2013).
In studies (Ermakova et al., 2013; Menshova et al., 2014) the an-
ticancer effect of laminaran fractions from Eisenia bicyclis was in-
vestigated. The laminaran from this alga had an unusual structure and
was a complex, branched high-molecular-weight 1,3;1,6-β-D-glucan
with a significant amount of 1,6-linked galactose residues (ratio of
bonds 1,3:1,6 = 3:2). It was shown that decreasing the molecular
weight of native laminaran to a determined limit and increasing the
content of 1,6-linked glucose residues increased the anticancer effect.
Recently, the in vitro anticancer activity of the laminarans isolated
from the brown seaweeds S. cichorioides, S. japonica and F. evanescens
and their chemically sulfated derivatives was demonstrated through
evaluation of their efficacy in inhibiting the proliferation, colony for-
mation and migration of human colorectal adenocarcinoma, malignant
melanoma and breast adenocarcinoma cells (Malyarenko et al., 2017).
The results revealed that the sulfation of laminarans increased their
anticancer effect. In the present work, we showed that inhibiting ac-
tivity of sulfated laminarans from Dictyota dichotoma was the highest
compared with that of native laminaran. We also proposed that it was
related to the degree of sulfation.
Next, we studied the effect of the investigated polysaccharides on
colony formation cancer cells after irradiation. DLD-1 cells were chosen
because the effect of the investigated polysaccharides on colony for-
mation of this cell line was highest. We developed conditions for irra-
diation of DLD-1 cells. The number of colonies was reduced by 8, 18
and 23% compared with the control at radiation doses of 4, 6 and 8 Gy,
respectively (Fig. 4A). We choose a dose of 4 Gy to study the effect of
the investigated polysaccharides on colony formation cancer cells after
irradiation. First we determined the effect of the polysaccharides DdL,
DdLs and DdF at a concentration of 40 μg/mL on colony formation of
DLD-1 human cancer cells using the soft agar clonogenic assay
(Fig. 4B). DdL, DdLs and DdF at 40 μg/mL did not inhibit this process
(data not shown). However,it was shown that the investigated poly-
saccharides dramatically decreased the number and size of cancer cells
colonies irradiated with 4 Gy. Fucoidan decreased the number and size
of DLD-1 cells colonies at 7 and 14% compared with cells irradiated
with 4 Gy, respectively. DdL decreased the number and size of DLD-1
Fig. 3. Inhibitory effect of native (DdF, DdL) and modified (DdLs) polysaccharides from
D. dichotoma against colony formation of human colon cancer cells HCT-116, HT-29, and
DLD-1.
DdLs prevented the formation and growth of colonies of these human
cancer cells at 35%.
The brown algae Saccharina cichorioides produced polysaccharides,
the structure of which was well characterized. The fucoidan from S.
cichorioides was fucan with a main chain of 1,3-linked α-L-fucose re-
sidues and a small number of 1,4-linked fucose residues. A non-sig-
nificant number of single α-L-fucose residues were in the branches at
the C2 position. Sulfate groups occupied positions 2 and 4 of the fu-
copyranose residues (Anastyuk et al., 2010; Anastyuk et al., 2017;
Zvyagintseva et al., 2003). The laminaran from S. cichorioides was
glucan with a main chain of 1,3-linked glucose residues. Single glucose
residues were found on the branches at the C6 position (ratio of bonds
1,3:1,6 = 9:1) (Zvyagintseva et al., 1994).
The effect of fucoidan and laminaran from the brown alga S. ci-
choriodes was previously demonstrated by our group. The fucoidan was
showed to inhibit colony formation of human colon cancer DLD-1 cells
by 75% (Vishchuk, Ermakova, & Zvyagintseva, 2013). The laminaran
25

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CarbohydratePolymers180(2018)21–28
Fig. 4. Effect of radiation and native (DdF, DdL) and modified (DdLs) polysaccharides from D. dichotoma against colony formation of human colon cancer cells DLD-1. (A) Effect of
different doses of radiation on colony formation of DLD-1cells. (B) Synergistic effect of DdF, DdL and DdLs with radiation (4 Gy) against amount and size of colony formation of DLD-
1cells.
26

R.V. Usoltseva et al.
CarbohydratePolymers180(2018)21–28
2106–2130.
cells colonies by 24 and 18%, respectively, whereas DdLs resulted in
corresponding decreases of – at 46 and 23%, respectively. This finding
is very important, because during irradiation of the human body,
normal cells are also exposed to irradiation. Polysaccharide can in-
crease the effect of irradiation on cancer cells and safeguard the sur-
vival of normal cells. It was demonstrated for the first time that fu-
coidan, laminaran and sulfated derived of laminaran inhibited not only
the number of irradiated cell colonies but also their size.
Anastyuk, S. D., Shevchenko, N. M., Nazarenko, E. L., Imbs, T. I., Gorbach, V. I.,
Dmitrenok, P. S., et al. (2010). Structural analysis of a highly sulfated fucan from the
brown alga Laminaria cichorioides by tandem MALDI and ESI mass-spectrometry.
Carbohydrate Research, 345, 2206–2212.
Anastyuk, S. D., Shevchenko, N. M., Usoltseva (Menshova), R. V., Silchenko, A. S.,
Zadorozhny, P. A., Dmitrenok, P. S., et al. (2017). Structural features and anticancer
activity in vitro of fucoidan derivatives from brown alga Saccharina cichorioides.
Carbohydrate Polymers, 157, 1503–1510.
Bradford, M. M. (1976). Rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Analitical
Biochemistry, 72, 248–254.
Byon, Y. Y., Kim, M. H., Yoo, E. S., Hwang, K. K., Jee, Y., Shin, T., et al. (2008).
Radioprotective effects of fucoidan on bone marrow cells: Improvement of the cell
survival and immunoreactivity. Journal of Veterinary Science, 9, 359–365.
Camara, R. B. G., Costa, L. S., Fidelis, G. P., Nobre, L. T. D. B., Dantas-Santos, N., Cordeiro,
S. L., et al. (2011). Heterofucans from the brown seaweed Canistrocarpus cervicornis
with anticoagulant and antioxidant activities. Marine Drugs, 9, 124–138.
Dodgson, K. S. (1961). Determination of inorganic sulphate in studies on the enzymic and
non-enzymic hydrolysis of carbohydrate and other sulphate esters. Biochemical
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Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric
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4. Conclusion
In the results of this investigation, the highly purified laminaran
DdL and fucoidan DdF were obtained from the brown alga Dictyota
dichotoma. Fucoidan was determined to be a sulfated (28.9%) and
acetylated heteropolysaccharide containing fucose, galactose, mannose
and glucose residues (57.9, 20.4, 12.4 and 9.2 mol%, respectively).
Laminaran was 1,3;1,6-β-D-glucan with a 1,3:1,6 = 3:1 bond ratio. The
structure of DdL was investigated by 2D spectroscopy NMR; laminaran
contained predominantly repeating structural units of tetrasaccharide
with three 1,3-linked β-D-glucopyranose residues and branch of single
glucose at C6.
The structure of DdL was modified by sulfation to obtain the sul-
fated (43.7%) laminaran derivative DdLs. Sulfate groups were identi-
fied at C2, C4 and C6 of glucose residues.
The anticancer effect of three obtained polysaccharides DdF, DdL
and DdLs was studied in vitro at a concentration of 200 μg/mL on
human colon cancer cells HCT-116, HT-29 and DLD-1. The native la-
minaran had little anticancer effect on all cell lines. Sulfated poly-
saccharides showed slight inhibition of colony formation of HCT-116
and HT-29 cells and powerful inhibition of DLD-1 cells (46 and 35% for
DdF and DdLs, respectively).
Ermakova, S., Men’shova, R., Vishchuk, O., Kim, S. M., Um, B. H., Isakov, V., et al. (2013).
Water-soluble polysaccharides from the brown algae Eisenia bicyclis: Structural
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laminarin. Experimental and Therapeutic Medicine, 6, 1259–1264.
We firstly investigated the effect of DdF, DdL and DdLs (40 μg/mL)
against cells of DLD-1 cancer cells after irradiation. No investigated
polysaccharides showed anticancer activity at this concentration, but
they decreased the amount (7, 24 and 46% for DdF, DdL, and DdLs,
respectively) and size (14, 18 and 23% for DdF, DdL, and DdLs, re-
spectively) of colony of cancer cells irradiated with 4 Gy.
We have demonstrated the in vitro anticancer activity of poly-
saccharides from the brown alga Dictyota dichotoma through evaluation
of their inhibitory activity of colony formation of human colorectal
adenocarcinoma cells. The current study showed that fucoidan, lami-
naran and sulfated laminaran showed a synergistic effect with X-ray
radiation against cancer cells. Thus, the application of the investigated
polysaccharides as additional therapy during radiotherapy can provide
a possible strong effect with a low dose of radiation.
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Polysaccharides from the brown seaweed Padina tetrastromatica: Characterization of a
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Structural characterization of β–D–(1 → 3,1 → 6)–linked glucans using NMR spec-
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coglucuronan from the alga Spatoglossum schroederi. Plant Science, 132, 215–228.
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Santos, N., et al. (2011). Anticoagulant, antioxidant and antitumor activities of het-
erofucans from the seaweed Dictyopteris delicatula. International Journal of Molecular
Sciences, 12, 3352–3365.
Acknowledgments
Malyarenko, O. S., Usoltseva, R. V., Shevchenko, N. M., Isakov, V. V., Zvyagintseva, T. N.,
& Ermakova, S. P. (2017). In vitro anticancer activity of the laminarans from Far
Eastern brown seaweeds and their sulfated derivatives. Journal of Applied Phycology,
29, 543–553.
The part of this work related to isolation and structural elucidation
of polysaccharides was supported mainly by the Russian Foundation for
Basic Research (16-33-60023), and the investigation of biological ac-
tivity of obtained polysaccharides was predominantly funded by the
Russian Science Foundation (16-14-10131). We thank Dr. Isakov V.V.,
PIBOC FEB RAS for his help with NMR spectra of polysaccharides.
Medeiros, V. P., Queiroz, K. C. S., Cardoso, M. L., Monteiro, G. R. G., Oliveira, F. W.,
Chavante, S. F., et al. (2008). Sulfated galactofucan from Lobophora variegata:
Anticoagulant and anti-inflammatory properties. Biochemistry, 73, 1018–1024.
Men'shova, R. V., Ermakova, S. P., Rachidi, S. M., Al-Hajje, A. H., Zvyagintseva, T. N., &
Kanaan, H. M. (2012). Seasonal variations of the composition, structuralfeatures, and
antitumor properties of polysaccharides from Padina pavonica (Lebanon) as a func-
tion of composition. Chemistry of Natural Compounds, 47, 870–875.
Menshova, R. V., Ermakova, S. P., Anastyuk, S. D., Isakov, V. V., Dubrovskaya, Y. V.,
Kusaykin, M. I., et al. (2014). Structure, enzymatic transformation and anticancer
activity of branched high molecular weight laminaran from brown alga Eisenia bi-
cyclis. Carbohydrate Polymers, 99, 101–109.
Park, H., Kim, I., Kim, J., & Nam, T. (2012). Induction of apoptosis by laminarin, reg-
ulating the insulin-like growth factor-IR signaling pathways in HT-29 human colon
cells. International Journal of Molecular Medicine, 30, 734–738.
Park, H., Kim, H., Kim, J., & Nam, T. (2013). Induction of apoptosis and the regulation of
ErbB signaling by laminarin in HT-29 human colon cancer cells. International Journal
of Molecular Medicine, 32, 291–295.
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