Food Hydrocolloids 42 (2014) 280e288

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Isolation and antioxidant capacity of fucoidan from selected Malaysian

seaweeds
Seng Joe Lim a, Wan Mustapha Wan Aida a, *, Mohamad Yusof Maskat a, Said Mamot a,
Jokiman Ropien b, Diah Mazita Mohd b
a

School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor,
Malaysia
Cosmetics and Natural Products Programme, Industrial Biotechnology Research Centre, SIRIM Berhad, No. 1, Persiaran Dato Menteri, Seksyen 2,
Peti Surat 7035, 40911 Shah Alam, Selangor, Malaysia
b

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 3 January 2013
Accepted 10 March 2014
Available online 18 March 2014

The objective of this research was to screen and isolate fucoidan from Malaysian seaweeds and subsequently determine its antioxidant capacity. A screening test employing a colorimetric method was
conducted on ve types of Malaysian brown and red seaweeds. It was found that Sargassum binderi
contained the highest fucoidan content (6.16
0.08%). Thus, fucoidan was isolated from S. binderi (yield
7.5%, purity 89.63%) for the determination of its antioxidant capacity. The isolated fucoidan was identied
using high performance anion exchange chromatography (HPAEC) and attenuated total reectance
Fourier transform infra-red (ATR-FTIR) spectroscopy. The antioxidant assays performed were total
phenolic content (TPC), free-radical scavenging activity (DPPH), reducing power, superoxide anion
scavenging activity (SOA) and hydroxyl radical scavenging activity (OH). The antioxidant capacity of the
extracted fucoidan (Fsar) was compared with those of a commercial food-grade fucoidan (Fysk) and of
BHA, BHT and ascorbic acid. All the antioxidant assays performed showed either Fsar has signicantly
higher (p < 0.05) or do not differ signicantly (p > 0.05) in activities compared to that of Fysk. At the same
time, both Fsar and Fysk showed signicant (p < 0.05) antioxidant capacity in terms of superoxide anion
and hydroxyl radical scavenging activities compared to those of the synthetic antioxidants. This shows
that Fsar has the potential to be commercialised as a functional food product or as bioingredients with
high antioxidative properties.
2014 Elsevier Ltd. All rights reserved.

Keywords:
Antioxidant
Attenuated total reectance Fourier
transform infra-red (ATR-FTIR)
Fucoidan
Malaysian seaweeds
Sargassum binderi

1. Introduction
Marine resources, in particular seaweeds, have drawn signicant attention in recent years in the search for bioactive compounds to develop as functional food and nutraceutical products
(Souza et al., 2012). The seaweed industry has an annual global
value of USD 5.5e6 billion and is mainly used for food (USD 5
billion), phycocolloids (hydrocolloids), fertiliser, animal feed additives, cosmetics and medicines (Phang, 2010). The Food and Agricultural Organization of the United Nations (FAO, 2012) reported
that in 2010, worldwide aquatic plant production reached 19
million tonnes, worth USD 5.56 billion. In Malaysia, aquatic plant
production has developed enormously over the past few years,
from 18,863 tonnes worth USD 1.99 million in 2001 to 207,892

* Corresponding author. Tel.: 603 89213870; fax: 603 89213232.

E-mail addresses: wawm@ukm.my, wanaidawm@gmail.com (W.M. Wan Aida).
http://dx.doi.org/10.1016/j.foodhyd.2014.03.007
0268-005X/ 2014 Elsevier Ltd. All rights reserved.

tonnes worth USD 17.44 million in 2010. The value of aquatic plants
in Malaysia has also increased tremendously, from USD 56.78 per
tonne in 2009 to USD 83.91 per tonne in 2010 (FAO, 2012).
Malaysian national frameworks and strategies have placed a signicant emphasis on seaweed production. This includes the Ninth
Malaysia Plan (2006e2010), the Third National Agricultural Policy
(1998e2010), the recent adoption of the National Aquaculture
Centre and the Malaysian Budget 2010, with seaweed being
mentioned specically as one of the most important food farming
commodities for the country (Kaur & Ang, 2009). The abundant
supply of seaweeds in Malaysia offers great opportunities for producing and extracting several functional ingredients, such as
fucoidan, alginate, agar and carrageenan.
Seaweeds are marine macroalgae that can be classied into the
Chlorophyta (green seaweeds), Rhodophyta (red seaweeds) and
Phaeophyta (brown seaweeds) (Phang, 2010). Fucoidan is a type of
glyconutrient and one of the main polysaccharides of brown seaweeds. According to Bilan et al. (2010) and Zvyagintseva et al. (1999),

S.J. Lim et al. / Food Hydrocolloids 42 (2014) 280e288

fucoidan is the family of sulphated homo and heteropolysaccharides

composed mainly of a-(1 / 2)-, a-(1 / 3)- and/or a-(1 / 4)-linked
a-L-fucosepyranose residues. The main monomer in fucoidan is
fucose, one of the eight essential biological sugars. It also contains
galactose, mannose, xylose and glucuronic acid residues (Bilan et al.
2010). Fucoidan is a nontoxic polyelectrolyte that possesses various
pharmacological activities, i.e. antioxidant, antibacterial, antiviral,
antitumour and anticoagulant activities, and it has also been
developed as a specialised type of nutraceutical and food supplement (Bilan et al., 2002; Zvyagintseva et al., 1999). Brown seaweed
extracts (fucoidan) and seaweed polysaccharides are reported to
have strong antioxidative properties and have high potential to be
applied in pharmaceuticals, nutraceuticals, cosmeceuticals and
functional food (Barahona, Chanda, Encinas, Matsuhiro, & Ziga,
2011; Li, Lu, Wei, & Zhao, 2008; Wijesinghe & Jeon, 2012). Due to
concerns regarding the carcinogenic and toxic effects of synthetic
antioxidants, the demand for natural antioxidants has increased
(Yangthong, Towatana, & Phromkunthong, 2009).
Despite the great number of studies on fucoidan, none has
investigated fucoidan from Malaysian seaweeds. It is well known
that the biological activities of fucoidan vary according to the season, the age of the population, species and geographic location
(Rioux, Turgeon, & Beaulieu, 2007; Zvyagintseva et al., 2003). The
abundant supply of Malaysian seaweeds and the large variety of
their species make it possible to exploit Malaysian seaweeds for
fucoidan production. In this research, ve types of Malaysian
seaweed were screened using a colorimetric method to determine
which type had the highest fucoidan content; based on this analysis, Sargassum binderi was then chosen for further analysis.
Fucoidan was extracted from S. binderi and was then isolated and
lyophilised to obtain fucoidan in solid form (Fsar). The isolated
fucoidan was then tested for its antioxidative activities, in which
commercial food-grade fucoidan (Fysk) and synthetic antioxidants
(BHA, BHT and ascorbic acid) were used as comparisons.
2. Materials and methods
2.1. Materials and samples
Brown seaweeds (S. binderi and Padina sp.) and red seaweeds
(Gracilaria sp., Eucheuma cottonii and Eucheuma spinosum) were
obtained from Semporna, Sabah, Malaysia. All the seaweeds were
obtained in November 2010. Commercial food-grade fucoidan with
92% purity from Okinawa mozuku was supplied by Yaizu Suisankagaku Industry Co., Ltd., Yaizu City, Japan (Fysk). Fucoidan
standard (from Fucus vesiculosus) was obtained from Sigma, UK
(Fsig). Synthetic antioxidant butylated hydroxyanisole (BHT)
(Merck, Germany), butylated hydroxytoluene (BHT) (Sigma, UK)
and ascorbic acid (Fluka, UK) were also used. All other chemicals
were of analytical grade and purchased from Sigma, unless otherwise stated.
2.2. Sample preparation
Fresh seaweed samples were washed to remove salt and impurities and dried at 40  C in an oven (Protech, Texas, USA) for 72 h.
They were then cut into small pieces with a knife mill before being
ground into powder form using a high-speed grinder (Kuao Fung
Electronic and Machine, Taiwan). The samples were kept at 4  C
until use.
2.3. Screening of fucoidan content
Fucoidan was extracted twice from 150 mg of all ve types of
seaweed samples (Brown seaweeds e S. binderi and Padina sp.; and

281

red seaweeds e Gracilaria sp., E. cottonii and E. spinosum) using

50 ml of 0.2 N HCl at 70  C for 1 h with mechanical stirring (Usov,
Smrnova, & Klochkova, 2001). The residues were separated through
centrifugation (Eppendorf Centrifuge 5810R, at 3500g, 5 min), and
the supernatants were pooled and brought to 100 ml. Aliquots of
1 ml were pipetted into test tubes placed in an ice-water bath, and
4.5 ml diluted sulphuric acid was added (1:6, H2O:H2SO4). After
1 min of cooling in an ice-water bath, the test tubes were placed
into a boiling-water bath for exactly 10 min. The aliquots were
allowed to cool to room temperature before adding 0.1 ml of 3% Lcysteine, and the solutions were mixed and allowed to stand for
30 min. The absorbance at 396 nm and 427 nm was measured in a
96-well plate using a BioTek Epoch microplate spectrophotometer
(Vermont, USA) (Hellebust & Craigie, 1978). A calibration curve
within the 0.0025e0.015% (w/v) range of fucoidan (Fysk) was used
to determine the fucoidan content in the samples. An n 3 replication was conducted in the screening process.
2.4. Isolation of fucoidan
The isolation of fucoidan was performed according to the
procedures reported by Bilan et al. (2004). The ground S. binderi
(50.0 g) was treated with 200 ml of MeOH:CHCl3:H2O (4:2:1) solution at room temperature with stirring for 24 h to extract/
remove lipids, coloured matter and low molecular weight components from the sample. The suspension was then ltered, and
the residue (ground S. binderi) was collected. This was performed a
few successive times until a clear ltrate was obtained at the end
of the treatment. The defatted seaweed (44.5 g) was then dried in
a vacuum oven (Vacucell MMM, Munich, Germany) at 50  C for
24 h. Extraction of fucoidan from S. binderi was performed using a
2% CaCl2 solution at 85  C with automated mechanical stirring
(200 rpm) for 24 h. This step was repeated six times to ensure
complete fucoidan extraction. The extracts were combined and
added with 10% hexadecyltrimethylammonium bromide solution
(in excess) to precipitate the fucoidan. The precipitate formed was
centrifuged (Eppendorf Centrifuge 5810R, 3000g, 10 min) and
rinsed
with
water
to
remove
excess
hexadecyltrimethylammonium bromide before being stirred in 20% ethanolic sodium iodide (Fisher Scientic) solution (5  100 ml) for 4e
5 days, rinsed with ethanol to remove excess sodium iodide, and
dissolved in water. The fucoidan solution was then dialysed
against distilled water for 4e5 days using a Visking tube with a
molecular weight cut-off (MWCO) of 2000 Da, before being
lyophilised to obtain fucoidan as a sodium salt in solid form (Fsar)
(Bilan et al., 2004).
2.5. Identication and purity of fucoidan
2.5.1. High performance anion exchange chromatography (HPAEC)
Fucoidan identication and purity determination were achieved
using high performance anion exchange chromatography (HPAEC)
with a Waters system (Massachusetts, USA) equipped with an auto
injector as performed by Rioux et al. (2007), with modications to
the temperature and ow rate parameters. The anion exchange
column used was Phenomenex Rezex RPM monosaccharide,
100  7.8 mm, with a guard column (Phenomenex 50  7.8 mm).
The isolated fucoidan (Fsar) and commercial food-grade fucoidan
(Fysk) were prepared at 5 mg/ml concentrations, and 20 ml was
injected into the HPAEC system. The elution was conducted using
deionised water at a ow rate of 0.2 ml/min and a temperature of
85  C. The eluent was detected using an evaporative light scattering
detector (Polymer Laboratories, PL-ELS 1000) with an evaporator
temperature of 120  C, a nebuliser temperature of 90  C, a transfer
line temperature of 30  C and a gas ow rate of 1.2 slm. The Waters

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S.J. Lim et al. / Food Hydrocolloids 42 (2014) 280e288

system works with Breeze software. A fucoidan standard (Fsig) was

used for the identication, while the determination of purity was
achieved using Fysk (92% purity) to provide a standard curve. A
factor of 92% was used in the calculation of purity of Fsar.
2.5.2. Attenuated total reectance Fourier transform infra-red (ATRFTIR) spectroscopy
Attenuated total reectance Fourier transform infra-red (ATRFTIR) spectroscopy was performed using a Perkin Elmer Precisely,
Spectrum 400, FT-IR/FT-NIR spectrometer (Massachusetts, USA)
tted with a Spectrum Universal ATR Accessory. A total of 4 scans
were performed on the samples with a scanning range of 4000e
650 cm1 and a resolution of 4 cm1 (Rodriguez-Jasso, Mussatto,
Pastrana, Aguilar, & Teixeira, 2011; Souza et al., 2012). The vibrational spectra were collected and analysed using Essential FTIR
1.1.0.0 software.
2.6. Determination of antioxidant capacity
2.6.1. Total phenolic content (TPC)
The TPC of fucoidan was measured using the FolineCiocalteu
method as described by Chew, Lim, Omar, and Khoo (2008). Samples of Fsar and Fysk were prepared at 2 mg/ml. The samples (0.3 ml)
were mixed with 1.5 ml FolineCiocalteu reagent and 1.2 ml sodium
carbonate solution (7.5% w/v). The reaction mixtures were incubated in the dark for 90 min before measuring the absorbance at
765 nm in a 96-well plate (200 ml) using a BioTek Epoch microplate
spectrophotometer. A standard curve of gallic acid with a concentration range of 5e50 ppm was prepared, and the TPC was
expressed as gallic acid equivalents per 100 g sample (GAE/100 g).
The TPC of Fsar and Fysk were compared with those of BHA, BHT and
ascorbic acid at the same concentration. An n 3 replication was
conducted on the total phenolic content.
2.6.2. Free-radical scavenging assay (DPPH)
Free scavenging activity was measured based on the scavenging
ability of stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals by
Fsar and Fysk as described by Chew et al. (2008). A series of Fsar and
Fysk concentrations (0.05e0.5% w/v) were prepared, and 1 ml of
each Fsar and Fysk concentration was mixed with 2.9 ml methanolic
0.15 mM DPPH. After incubation of the reaction mixture in the dark
for 30 min, the absorbance was measured at 517 nm in a 96-well
plate (200 ml) using a BioTek Epoch microplate spectrophotometer.
The DPPH tests were performed in triplicate. The IC50 of Fsar and Fysk
were calculated and compared with that of BHA, BHT and ascorbic
acid using the formula below:

% Free radical scavenging activity A1  A2 =A1   100

(1)

where A1 is the absorbance of the DPPH blank and A2 is the

absorbance of the sample with DPPH.
The DPPH radical scavenging was then expressed in terms of
ascorbic acid equivalent antioxidant capacity (AEAC), which was
calculated based on the equivalent antioxidant capacity of ascorbic
acid with 100 g of sample as follows:

.
i
h
AEACmg AA=100 g IC50AA IC50sample  100; 000

(2)

prepared at 2 mg/ml, were mixed with phosphate buffer (2.5 ml,

0.1 M, pH 6.6) and potassium ferricyanide [K3Fe(CN)6] (2.5 ml, 1%
w/v). The solutions were then incubated at 50  C for 20 min. Trichloroacetic acid (2.5 ml, 10%) was added to the solutions, and the
mixtures were centrifuged at 2000g for 10 min (Eppendorf
Centrifuge 5810R). A portion of the samples upper layers (2.5 ml)
were mixed with 2.5 ml distilled water and 0.5 ml 0.1% (w/v) FeCl3
and were left at room temperature for 30 min before measuring the
absorbance at 700 nm in a 96-well plate (200 ml) using a BioTek
Epoch microplate spectrophotometer. The reducing power assay
was performed in triplicate. The results were expressed as gallic
acid equivalents (mg GAE/100 g) and compared with BHA, BHT and
ascorbic acid (2 mg/ml).

2.6.4. Superoxide anion (SOA) scavenging activity

The superoxide anion scavenging activity was determined by
measuring the inhibition of pyrogallol auto-oxidation (Heo, Park,
Lee, & Jeon, 2005; Marklund & Marklund, 1974). Both Fsar and Fysk
were prepared at 2 mg/ml. The samples (0.3 ml) were mixed with
2.6 ml phosphate buffer (50 mM, pH 8.24) and 90 ml of 3 mM
pyrogallol solution (dissolved in 10 mM HCl). The absorbance,
which reects the inhibition rate of pyrogallol auto-oxidation,
was measured at 325 nm in a 96-well plate (200 ml) using a
BioTek Epoch microplate spectrophotometer in which the absorbance was recorded every 1 min interval for 10 min. A replication
of n 3 was performed in the superoxide anion scavenging activity test. The percentages of superoxide anion scavenging activities of the samples were compared with those of BHA, BHT
and ascorbic acid (2 mg/ml) and were calculated using the
following equation:

Scavenging rate% 1  A2  A1 =A0   100

(3)

where A1 is the absorbance of the sample at 0 min, A2 is the

absorbance of the sample at the 10th min, and A0 is the autoxidation rate of pyrogallol for the blank (the change of absorbance in the
blank from 0 min to 10 min).

2.6.5. Hydroxyl radical scavenging activity (OH)

The hydroxyl radical scavenging activity (OH) was determined
according to Zhao et al. (2012). Both Fsar and Fysk were prepared at
2 mg/ml. The hydroxyl radical was generated using the Fenton reaction, where 0.5 ml of 9 mM ferrous sulphate (FeSO4) solution was
added to 1.0 ml of 8.8 mM hydrogen peroxide (H2O2) (35% purity,
HmbG Chemicals) solution. This mixture was then added to the Fsar
and Fysk samples prepared earlier, before adding 0.2 ml of 9 mM
salicylic acid solution. Another set of reaction solutions was prepared as above without the addition of salicylic acid. The mixtures
were allowed to stand at 37  C for 1 h before the absorbance was
measured at 510 nm in a 96-well plate (200 ml) using the BioTek
Epoch microplate spectrophotometer. The hydroxyl radical scavenging activities of the samples were compared with those of BHA,
BHT and ascorbic acid at the same concentration (2 mg/ml). The
hydroxyl radical scavenging activity was determined in triplicate,
and the activity was calculated as follows:

Hydroxyl radical scavenging rate%

where AA is ascorbic acid and IC50 is the inhibition concentration at
50%.
2.6.3. Reducing power assay
The reducing power assay was performed according to Kumar,
Ganesan, and Rao (2008). Both the Fsar and Fysk, which were

1  fA1  A2 =A0 g  100

(4)

where A0 is the absorbance of the blank (without sample), A1 is the

absorbance of the reaction containing the sample and salicylic acid,
and A2 is the absorbance of the reaction containing the sample but
without salicylic acid.

S.J. Lim et al. / Food Hydrocolloids 42 (2014) 280e288

2.7. Statistical analysis

Analysis was performed in triplicate (n 3), except for the
HPAEC and FTIR analyses, which were done once. Data were obtained as the mean and standard deviation and analysed using oneway ANOVA followed by Duncans multiple range test (DMRT) using SAS version 6.12 for Windows. The difference in mean values
was considered signicant when p < 0.05.
3. Results and discussion
3.1. Screening and isolation of fucoidan content
The screening of the fucoidan content was performed using a
colorimetric method in which two different wavelengths, 396 nm
(l396) and 427 nm (l427), were used to determine the fucose content
via the specic colour reactions of fucose with L-cysteine and sulphuric acid. The difference in absorbance between the two wavelengths (Dl396  l427) differentiates fucose from other hexoses
present, as the absorbance of fucose has a maximum at l396 but is
near zero at l427. At the same time, the absorbance of all other
hexoses is the same at both wavelengths of l396 and l427 (Dische &
Shettles, 1948; Usov et al., 2001). This test acted as the preconrmation of fucoidan presence in the seaweeds.
As shown in Table 1, the brown seaweeds, i.e. S. binderi
(6.16
0.08%) and Padina sp. (2.06
0.23%), showed the presence
of fucoidan at signicantly higher (p < 0.05) amounts compared to
those in the red seaweeds. The low readings from the red seaweeds
might not be from fucoidan, as explained by Usov et al. (2001),
where the fucose detected in red seaweeds was actually the
product of a colour reaction from other 6-deoxyhexoses found in
seaweed. However, as a rule, brown seaweeds do not contain these
compounds, and the colour reactions with other sugars are
excluded by the colour measurement at the two wavelengths (Usov
et al., 2001).
As discussed by Wijesinghe and Jeon (2012), it was found in this
pre-conrmation test that brown seaweeds are the main source of
fucoidan. Sulphated polysaccharides are among the most abundant
and broadly studied polysaccharides from non-animal origin, and
brown seaweeds are recognised as a major source of sulphated
polysaccharides (fucoidan) with various biological activities. Due to
the high fucoidan content in S. binderi in the former work, further
isolation work was performed on this particular species for the
determination of its antioxidant capacity. In the extraction and
isolation of fucoidan, a total of 3.34 g of fucoidan (Fsar) was successfully isolated from 44.5 g of S. binderi, giving a 7.5% yield.
Studies have been conducted on the fucoidan content of various
species of brown seaweed collected in different seasons and locations. These studies showed that the fucoidan content varies with
the species, season and location. A notable study by Usov et al.
(2001), who studied the fucoidan content of 17 species of brown
seaweed collected in July 1990 from Avancha Bay, Kamchatka,

Table 1
Fucoidan content of seaweed samples (n 3).
Sample
Brown seaweed
Sargassum binderi
Padina sp.
Red seaweed
Gracilaria sp.
Eucheuma spinosum
Eucheuma cottonii
aec

Fucoidan content (%)

6.16
0.08a
2.06
0.23b
0.27
0.08c
0.33
0.06c
0.36
0.10c

Different letters indicate a statistically difference (p < 0.05).

283

Russia, showed that the fucoidan content ranged from 0.4% (Desmarestia intermedia P. et R., Laminaria dentigera Kjellm, and
Arthrothamnus bidus P. et R.) to 20.4% (Saudersella simplex (Saund.)
Kylin).
Another study conducted by Zvyagintseva et al. (2003) showed
variations in the fucoidan content from brown seaweeds of
different species, seasons and locations, where samples were
collected from the Troitsa Bay (Laminaria cicorioides) and the
Rifovaya Bay (Laminaria japonica), Japan and from a few islands in
the Sea of Okhotsk, Russia (Fucus evanescens). These samples were
collected in different seasons (from August 1996 to August 1999). It
was shown that the fucoidan content in terms of dry weight basis
varied from 1.1% to 12.0% depending on the species, season and
location. A more recent study by Rioux, Turgeon, and Beaulieu
(2009), who collected brown seaweed (Saccharina longicruris)
from two locations, Perc and LAnse--Beauls, both in Qubec,
Canada, between May 2005 to June 2006, showed that the fucoidan
content from this species in different seasons and locations varied
from 1.6% to 4.5%.
All these studies concluded that brown seaweeds contain
fucoidan and that their content varies according to the species,
season and location. Therefore, this justies the study of fucoidan
extracted from Malaysian brown seaweed, S. binderi, as there are
differences in fucoidan from different sources. The fucoidan yield of
7.5% (Fsar) is acceptable, as it is within the range of fucoidan content
from the various studies discussed above.
3.2. Identication of fucoidan
3.2.1. High performance anion exchange chromatography (HPAEC)
An elution using high performance anion exchange chromatography (HPAEC) was performed on the Fsar and Fysk, which were
compared to the fucoidan standard (Fsig). This acted as a primary
conrmation test. The chromatograms of the HPAEC (Fig. 1) show
that the retention time of Fsig was 10.537 min, while Fsar and Fysk
had retention times of 10.343 min and 10.632 min respectively. The
Breeze system matched all three chromatogram peaks as the same
compound; i.e. fucoidan. Therefore, the primary conrmation of
fucoidan was successfully achieved. The purity of Fsar was successfully determined at 89.63%, using Fysk to plot the standard curve
with an R2 0.9927.
3.2.2. Attenuated total reectance Fourier transform infra-red (ATRFTIR) spectroscopy
A secondary conrmation test on Fsar was then carried out using
ATR-FTIR spectroscopy. The ATR-FTIR spectra of both Fsar and Fysk
are shown in Fig. 2. This analysis was conducted to determine
whether Fsar and Fysk have similar infra-red absorption properties to
those identied in published data. Both the Fsar and Fysk spectra
showed similar signals, with two bands in the 4000e2000 cm1
region and numerous signals with similar wavenumbers within the
2000e800 cm1 region. Common to all polysaccharides and
seaweed polysaccharides, two bands appeared in the 4000e
2000 cm1 region of the ATR-FTIR spectra. There was a broad band
centred at 3378 cm1 (Fsar) and 3383 cm1 (Fysk), which was
assigned to hydrogen bonded OeH symmetrical and asymmetrical
stretching vibrations (Coimbra, Barros, Barros, Rutledge, &
Delgadillo, 1998; Gomez-Ordonez & Ruperez, 2011). These peaks
were also caused by the presence of moisture in the sample, as H2O
contains OeH bonds that give a stretching vibration signal
(Silverstein & Webster, 1998, pp. 71e111). There was another weak
signal at 2944 cm1 (Fsar) and 2941 cm1 (Fysk) due to CeH
stretching vibrations (Coimbra et al., 1998; Gomez-Ordonez &
Ruperez, 2011). Additionally, the medium to strong IR absorption
bands (1200e970 cm1) are mainly due to CeC and CeO stretching

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S.J. Lim et al. / Food Hydrocolloids 42 (2014) 280e288

Fig. 1. HPAEC chromatograms of (a) fucoidan standard (Fsig), (b) fucoidan isolated from Sargassum binderi (Fsar) and (c) commercial food-grade fucoidan (Fysk).

in pyranoid rings and to CeOeC stretching in glycosidic bonds.

Intense absorption in this spectral region is common for all polysaccharides (Synytsya et al., 2010).
The typical absorption bands of fucoidan are the bands that
indicate the presence of sulphate (SO4) and methyl (CH3) groups, as
fucoidan is a sulphated polysaccharide and contains mainly fucose,
a monosaccharide that has a methyl group attached to the C5 position (Ale, Mikkelsen, & Meyer, 2011; Li et al., 2008). The signals at
1420 cm1 (Fsar) and 1414 cm1 (Fysk) were attributed to the presence of the asymmetrical bending vibration of CH3, while the signals at 1369 cm1 (Fsar) and 1367 cm1 (Fysk) were assigned to the

symmetrical bending vibration of CH3 (Kim et al., 2010; Silverstein

& Webster, 1998).
There was a strong signal at 1025 cm1 (both Fsar and Fysk) and a
shoulder signal at 1076 cm1 (Fsar) and 1075 cm1 (Fysk), which
were assigned to the stretching vibrations of sulphoxides (SaO).
The strong signals for sulphoxides indicate that there were significant amounts of sulphate groups in the fucoidan (Marais &
Joseleau, 2001; Silverstein & Webster, 1998). A rather sharp signal
at 963 cm1 (Fsar) and 958 cm1 (Fysk) indicates the presence of the
asymmetrical stretching vibration of CeOeS bonds, while the signals at 822 cm1 (Fsar) and 837e849 cm1 (Fysk) were assigned to

S.J. Lim et al. / Food Hydrocolloids 42 (2014) 280e288

285

Fig. 2. Attenuated total reectance e Fourier transform infra-red (ATR-FTIR) spectra of (a) fucoidan isolated from Sargassum binderi (Fsar) and (b) commercial food-grade fucoidan
(Fysk). I: OeH stretching vibrations. II: CeH stretching vibrations. III: Presence of moisture. IV: CH3 asymmetrical bending vibrations. V: CH3 symmetrical bending vibrations. VI:
SaO stretching vibrations. VII: CeOeS asymmetrical stretching vibrations. VIII: CeOeS symmetrical stretching vibrations.

the symmetrical stretching vibrations of CeOeS bonds (Synytsya

et al., 2010). At the same time, the absorption at 822 cm1 (Fsar)
indicates the presence of sulphate groups at the equatorial C2 and
C3 positions, while the absorption at 837e849 cm1 (Fysk) was
attributed to the sulphate groups at the axial C4 position (Patankar,
Oehninger, Barnett, Williams, & Clark, 1993).
Therefore, as mentioned above, the presence of sulphate and
methyl groups as well as of CeOeS bonds, which are typical in
fucoidan ATR-FTIR spectra, enabled us to identify the polysaccharide extracted from S. binderi (Fsar) as fucoidan. The signals at
1619 cm1 (Fsar) and 1616 cm1 (Fysk) were due to the bending vibrations of HOH, which indicate the presence of moisture in the
samples (Silverstein & Webster, 1998). Another peak at 1732 cm1
appeared in the Fysk spectra, and this peak was due to the CaO
stretching vibrations of carboxylic esters (Gomez-Ordonez &
Ruperez, 2011; Silverstein & Webster, 1998). This peak was not as
evident in the Fsar spectra because it was masked by the stronger
HOH bending vibration.
3.3. Antioxidant assays
In this research, the free-radical scavenging activity (DPPH)
assay examines the primary antioxidant potential, while the superoxide anion (SOA) scavenging activity and hydroxyl radical
(OH) scavenging activity assays examine the secondary antioxidant potential. The reducing power assay, on the other hand, examines both primary and secondary antioxidant capacity. There are
generally two types of antioxidants, namely, primary and secondary antioxidants, as mentioned above. Primary antioxidants, also

referred to as chain-breaking antioxidants, function as free-radical

acceptors/scavengers and delay or inhibit the initiation step or
interrupt the propagation step of auto-oxidation. Secondary antioxidants, on the other hand, are classied as preventive antioxidants, which slow the rates of oxidation reactions through various
mechanisms. Secondary antioxidants act as chelators for pro-oxidants (metal ions), provide H to primary antioxidants, decompose
hydroperoxide to nonradical species, deactivate singlet oxygen,
absorb ultraviolet radiation or act as oxygen scavengers. Therefore,
the main difference between primary and secondary antioxidants is
that secondary antioxidants do not convert free radicals into stable
molecules (Wanasundara & Shahidi, 2005).
3.3.1. Total phenolic content
According to Table 2, the total phenolic content (TPC) of Fsar
(3.69
0.15 mg GAE/100 g) was not signicantly (p > 0.05) different
from that of Fysk (3.64
0.23 mg GAE/100 g), but both Fsar and Fysk had
signicantly (p < 0.05) lower TPC than BHA, BHT and ascorbic acid.
This was expected because fucoidan is not a phenolic compound
but rather an aliphatic polysaccharide with monosaccharides,
namely, fucose, galactose, xylose, glucose, mannose and glucuronic
acid, as its building blocks. In contrast, BHA, BHT and ascorbic acid
have aromatic (phenolic) structures and therefore gave high TPC
readings (Bilan et al. 2004).
3.3.2. Free-radical scavenging activity (DPPH)
Antioxidants that have free-radical scavenging activity can
reduce free radicals, such as DPPH radicals. DPPH is a stable radical
that possesses a nitrogen free radical with a maximum absorbance

286

S.J. Lim et al. / Food Hydrocolloids 42 (2014) 280e288

Table 2
Comparison of antioxidant activities of the fucoidan extracted from Sargassum binderi (Fsar), commercial food-grade fucoidan (Fysk), BHA, BHT and ascorbic acid (n 3).
Sample

TPC (mg GAE/100 g)

DPPH

Reducing power (mg GAE/100 g)

IC50 (mg/ml)
Fsar
Fysk
BHA
BHT
Ascorbic acid

3.69
3.64
172.94
101.43
451.75

0.15c
0.23c
13.26b
18.60b
79.05a

2.01
1.87
(5.96
(9.59
(3.49

0.29a
0.08a
0.01)  104b
0.05)  104b
0.19)  104b

SOA inhibition (%)

OH scavenging (%)

AEAC (mg AA/100 g)

17.64
2.82a
18.71
0.87a
e
e
e

0.60
0.26
63.61
32.41
42.26

0.08d
0.01d
0.27a
0.78c
0.27b

26.78
20.57
6.41
24.41
98.85

1.90b
1.48d
0.69e
1.27c
0.25a

60.95
40.22
63.75
35.71
99.97

0.69b
2.68c
1.57b
2.62d
0.15a

aee

Different letters in the same column indicate a statistically signicant difference (p < 0.05).

at 517 nm (Kumar et al., 2008; Yangthong et al., 2009). Due to the

simplicity and convenience of this assay, it is used widely in freeradical scavenging activity assessments (Williams, Cuvelier, &
Berset, 1995).
Based on the results shown in Table 2, it was found that the IC50
of Fsar was not signicantly (p > 0.05) different from that of Fysk, but
both Fsar and Fysk possessed signicantly (p < 0.05) higher IC50
values than BHA, BHT and ascorbic acid. A lower IC50 is favourable
because it indicates that a lower concentration is needed for 50%
inhibition of the DPPH free radical. The same observation was made
for the ascorbic acid equivalent antioxidant capacity (AEAC),
whereby both Fsar and Fysk were not signicantly (p > 0.05)
different. AEAC is the equivalent of the antioxidant capacity of
ascorbic acid (in mg) in 100 g of sample. This shows that the
extracted fucoidan (Fsar) has a lower free-radical scavenging activity than that of synthetic antioxidants. Therefore, it was prudent
to carry out further studies on its secondary antioxidant capacity.
3.3.3. Reducing power
In terms of reducing power, it was found that both Fsar and Fysk
did not show signicant (p > 0.05) differences, but again, both were
signicantly (p < 0.05) lower than that of BHA, BHT and ascorbic
acid (Table 2). This shows that Fsar and Fysk exhibited low reducing
power.
Antioxidants with reducing power are those that can act as
electron donors and can reduce the oxidised intermediates of lipid
peroxidation processes, allowing them to act as primary and secondary antioxidants (Chanda & Dave, 2009). Such antioxidants
react with potassium ferricyanide (Fe3) to form potassium ferrocyanide (Fe2), which then reacts with ferric chloride (Fe3) to form
ferrous complexes (Fe2) that have a maximum absorbance at
700 nm (Ferreira, Baptista, Boas, & Barros, 2007; Jayanthi & Lalitha,
2011). Therefore, based on the DPPH and reducing power test results, both Fsar and Fysk have low primary antioxidant activity.
3.3.4. Superoxide anion scavenging activity
Superoxide anion radicals are formed from cellular oxidation
reactions in organisms, including in humans. Although it is a relatively weak oxidant, it decomposes to produce stronger oxidative
species, such as hydrogen peroxide and hydroxyl radicals, through
dismutation and other types of reactions. It is also the source of the
free radicals formed in vivo. SOA radicals and its derivatives are celldamaging, causing damage to DNA and cell membranes. Therefore,
it is of great important to scavenge SOA radicals (Sarikurkcu, Tepe,
Semiz, & Solak, 2010; Yangthong et al., 2009).
In this assay, the results in Table 2 show that the superoxide
anion (SOA) scavenging activity of Fsar (26.78
1.90% inhibition)
was signicantly (p > 0.05) higher compared to that of Fysk
(20.57
1.48% inhibition), BHA (6.41
0.69% inhibition) and BHT
(24.41
1.27% inhibition). Ascorbic acid had the signicantly
highest superoxide anion scavenging activity among all the samples. The superoxide anion scavenging activity was measured using
the pyrogallol auto-oxidation system, and the results were

expressed as the inhibition rate of superoxide production. The

signicantly (p > 0.05) higher SOA scavenging activity of Fsar
compared to that of Fysk, BHA and BHT demonstrates the secondary antioxidant potential of Fsar.
3.3.5. Hydroxyl radical scavenging activity (OH)
The hydroxyl radical is the most reactive free radical and can be
formed from superoxide anions and hydrogen peroxides in the
presence of metal ions, such as copper and iron. Hydroxyl radicals
can cause damage to nearly all types of biomolecules, including
proteins, DNA, polyunsaturated fatty acids, and nucleic acids
(Aruoma, 1998). These damages accelerate the ageing process and
cause cancer and several diseases (Zhu, Zhou, & Qian, 2006).
Therefore, the removal or scavenging of hydroxyl radicals in the
human body is one of the most effective defences by the body.
As shown in Table 2, Fsar (60.95
0.69%) has signicantly higher
(p < 0.05) hydroxyl radical scavenging activity compared to Fysk
(40.22
2.68%) and BHT (35.71
2.62%). It was also found that the
hydroxyl radical scavenging activity of Fsar was not signicantly
different (p > 0.05) compared to that of BHA (63.75
1.57%).
Ascorbic acid had the highest (p < 0.05) hydroxyl radical scavenging activity compared to the rest of the samples.
The tests of both superoxide anion and hydroxyl radical scavenging activities showed that Fsar demonstrates effective secondary
antioxidant capacity. Therefore, the fucoidan extracted from
Malaysian brown seaweed, S. binderi, has the potential to be a
source of natural secondary antioxidants.
3.3.6. Comparison of antioxidant activities with those in previous
works
In this research, it was clearly established that Fsar showed lower
primary but higher secondary antioxidant activity when compared
to synthetic antioxidants. The relatively low primary antioxidant
activity indicates that Fsar does not scavenge free radicals effectively. In contrast, the high secondary antioxidant activity of Fsar
showed that it can scavenge or quench pro-oxidants, such as oxygen. Interestingly, Wanasundara and Shahidi (2005) reported that
secondary antioxidants also function as H-donors to primary antioxidants, allowing secondary antioxidants to regenerate primary
antioxidants. This is a form of synergistic effect between primary
and secondary antioxidants.
Various studies have been conducted on the antioxidant activity
of the fucoidan extracted from different species of brown seaweed.
Similar antioxidant activities were observed in all these previous
works, with the fucoidan demonstrating antioxidant activity at
different levels depending on the type of antioxidant assay. According to Wang, Zhang, Zhang, and Li (2008) and Wang, Zhang,
Zhang, Song, and Li (2010), who studied the antioxidant activity
of the fucoidan extracted from L. japonica (cultured in Shazikou,
Qingdao, China e August 2007 and March 2005), the IC50 of the
DPPH scavenging activity of the fucoidan was 3.7 mg/ml, which is
higher than that of the current work, at 2.01 mg/ml (a lower IC50 is
more favourable). The study also reported that the extracted

S.J. Lim et al. / Food Hydrocolloids 42 (2014) 280e288

fucoidan displayed high superoxide anion and hydroxyl radical

scavenging activities and that the superoxide anion scavenging
activity was higher than that of BHA.
Another study by Souza et al. (2007) demonstrated that the
fucoidan extracted from F. vesiculosus and Padina gymnospora
showed signicant superoxide anion and hydroxyl radical scavenging activities in terms of its IC50 values. The current work
showed that both the superoxide anion and hydroxyl radical
scavenging activities of Fsar are signicantly higher than (p < 0.05)
or do not differ signicantly (p > 0.0.5) from those of BHA and BHT.
In a review paper by Li et al. (2008), the authors stated that
fucoidan from various sources showed signicant inhibition of
superoxide anion and hydroxyl radical scavenging activities, while
a lower DPPH radical scavenging activity was observed.
A study by Kuda, Tsunekawa, Hishi, and Araki (2005) showed a
similar trend in antioxidant activity as that observed in the current
work. The study used the brown seaweed Scytosiphon lomentaria
harvested in Wajima, Ishikawa, Japan, in March 2003. It was
discovered that the reducing power and the DPPH scavenging activity of the crude fucoidan were relatively low compared to that of
ascorbic acid. While no particular dilutions were given, the concentrations of crude fucoidan used in the study were approximately
(based on the values from the graphs) 0.3e5 mg/ml (a stock solution
of 5 mg/ml was prepared). The IC50 of the DPPH scavenging activity
was not determined in this concentration range, which shows that
the fucoidan from the current work (Fsar) has better DPPH scavenging
activity (IC50 2.01 mg/ml). In the same study, it was reported that
crude fucoidan showed superoxide anion scavenging activity.
Therefore, the Fsar from the current work has potential applications as a natural antioxidant, especially in terms of secondary
antioxidants. The Fsar gave signicantly higher (p < 0.05) values or
no signicant difference (p > 0.05) compared with Fysk in all antioxidant assays performed in the current work. It was also found
that the antioxidant capacity of Fsar is similar or superior to that
found in previous studies.
4. Conclusions
Among the screened samples, S. binderi had the highest fucoidan content. The polysaccharide extracted from S. binderi (Fsar)
(yield 7.5%) was identied as fucoidan (purity 89.63%) through
HPAEC and ATR-FTIR spectroscopy methods and exhibited antioxidant capacity. It was found that Fsar showed lower primary antioxidant activity but signicantly higher secondary antioxidant
activity, namely, superoxide anion and hydroxyl radical scavenging
activity, compared to that of the synthetic antioxidants (BHA and
BHT). The antioxidant properties of Fsar were comparable to those
of commercial, food-grade fucoidan (Fysk), as both the superoxide
anion and hydroxyl radical scavenging activities of Fsar were
signicantly higher (p < 0.05) than that of Fysk, and all the other
antioxidant assays showed no signicant difference (p > 0.05) between Fsar and Fysk. In addition, Fsar was found to have a superior or
at least similar antioxidant capacity compared to that observed in
previous studies. Therefore, Malaysian brown seaweed (S. binderi)
is a good source of fucoidan that exhibits antioxidant activity and
has the potential to be commercialised as a natural antioxidant
bioingredient.
Acknowledgements
This research was funded by STGL-007-2010 and OUP-2012-128
grants and the MyBrain15 (MyPhD) Scholarship by the Ministry of
Higher Education, Malaysia. The authors would like to thank SIRIM
Bhd, Malaysia for supplying the seaweed samples, Yaizu Suisankagaku Industry Co., Ltd., Yaizu City, Japan for providing

287

commercial food-grade fucoidan and the School of Chemical Sciences and Food Technology, Faculty of Science and Technology,
Universiti Kebangsaan Malaysia, which provided all the facilities
necessary for this research.

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