LWT - Food Science and Technology 73 (2016) 602e608

Contents lists available at ScienceDirect

LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

Polysaccharides from Laminaria japonica: Structural characteristics

and antioxidant activity
Chun Cui a, Jianghong Lu a, Dongxiao Sun-Waterhouse a, Lixia Mu b, c, d, Weizheng Sun a,
Mouming Zhao a, Haifeng Zhao a, *
a
School of Food Science and Engineering, South China University of Technology, Guangzhou 510641, PR China
b
Sericultural & Agri-Food Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510610, PR China
c
Key Laboratory of Functional Foods, Ministry of Agriculture, Guangzhou 510610, PR China
d
Guangdong Key Laboratory of Agricultural Products Processing, Guangzhou 510610, PR China

a r t i c l e i n f o a b s t r a c t

Article history: In the present study, polysaccharides from Laminaria japonica (LJPA-P) were investigated for their
Received 2 November 2015 structural characteristics and antioxidant activity. Three acidic polysaccharides LJPA-P1, LJPA-P2 and
Received in revised form LJPA-P3 were obtained from Laminaria japonica by sequential hot water and citric acid extraction, and
9 June 2016
fractionation by DEAE-Sepharose fast flow chromatography and serial NaCl solution elutions. Results
Accepted 3 July 2016
showed that three acidic polysaccharides differed in their monosaccharide compositions, sulfation,
Available online 4 July 2016
glycosidic linkages, molecular weights and antioxidant activities. LJPA-P3 was unique in its high sulfate
group and galactose contents, molar ratio of galactose, mannose and fucose (26.1: 1.3: 1) and dominance
Keywords:
Polysaccharide
of 1,3- and 1-linked galactose, exhibiting remarkably high oxygen radical absorbance capacity (ORAC,
Laminaria japonica 1247.22 mmol Trolox/g) and ABTS radical scavenging activity (up to 70%), which was quite different from
Antioxidant activity those of LJPA-P1 and LJPA-P2. Therefore, brown seaweed Laminaria japonica represents a good source of
Glycosidic linkages polysaccharides with significant antioxidant activity.
Sulfate group © 2016 Published by Elsevier Ltd.

1. Introduction
Marine plants containing large amounts of bioactive com-
pounds, represent a promising resources for food and cosmetic
industries. Marine algae naturally possess protective mechanisms
mediated by antioxidative compounds and intrinsic enzymes to
resist the harsh surrounding environment. Laminaria japonica, a
brown alga, is very popular in East Asia due to its unique flavor,
€ger, & Staerk,
taste and biological activities (Liu, Kongstad, Wiese, Ja
2016). The polysaccharides extracted from Laminaria japonica have
attracted more and more attention because of their multiple bio-
activities including antioxidative, anticoagulant, antivirus and
immune-modulating effects (Peng, Liu, Fang, Wu, & Zhang, 2012;
Zhang & Row, 2015).
Hot water extraction, one traditional method for natural poly-
saccharide production, usually requires a combined use of other
processes such as hydrolysis, chemical derivatization or physical
treatments, in order to obtain polysaccharides with specific
* Corresponding author.
E-mail address: hfzhao@scut.edu.cn (H. Zhao).
structure characteristics for higher bioactivities (Choi, Kim, & Lee,
2011; Liu, Wang, & Liu, 2016; Zeid, Aboutabl, Sleem, & El-Rafie,
2014). A novel antioxidative polysaccharide from Laminaria
japonica contained galactose, mannose and fucose in a molar ratio
of 9:2:2 was firstly prepared by mild acid hydrolysis (Xue et al.,
2001). Choi et al. (2011) found that low molecular weight
(6e9 kDa) and b-1,3- to b-1,6-linkages could significantly enhance
the biological activities of laminarin. Delattre, Fenoradosoa, and
Michaud (2011) pointed out that galactans, especially sulfate gal-
actans, had a positive impact on the biological activities of poly-
saccharides. Sun, Wang, Shi, and Ma (2009) revealed that a
polysaccharide fraction from Porphyridium cruentum containing
high sulfate group content (17.34%) exhibited very high antioxidant
activity. In our previous study, the Laminaria japonica poly-
saccharide obtained by citric acid extraction was found to have low
molecule weight and high antioxidant capacity (Lu, You, Lin, Zhao,
& Cui, 2013), whereas a directive process for producing a poly-
saccharide with the specific structural characteristics and antioxi-
dant activity was not proposed. Moreover, the relationships
between structural characteristics and antioxidant activity of
polysaccharides from Laminaria japonica have also not been fully

http://dx.doi.org/10.1016/j.lwt.2016.07.005
0023-6438/© 2016 Published by Elsevier Ltd.
 C. Cui et al. / LWT - Food Science and Technology 73 (2016) 602e608
elucidated.
Therefore, one objective of this study was to propose an
improved process for crude polysaccharide preparation from
Laminaria japonica by combining hot water extraction and citric
acid extraction. The second objective was to fractionate and purify
crude polysaccharide by DEAE-Sepharose fast flow chromatog-
raphy using water elution followed by increasing concentration of
NaCl elutions, and then to identify their chemical structures by gas
chromatography/mass spectrometry and Fourier infrared spectro-
photometry. The last objective was to evaluate antioxidant activity
of purified fractions of Laminaria japonica polysaccharide with ox-
ygen radical absorbance capacity (ORAC) and ABTS radical scav-
enging activity, and to elucidate structure-activity relationship of
the polysaccharide fractions.
2. Materials and methods
2.1. Materials and chemicals
Brown seaweed Laminaria japonica, collected on the coast of
Qingdao, Shandong, China in September 2014, was washed, sun-
dried and ground into a fine powder using a laboratory mill. The
powder particles that passed through a 40-mesh sieve were
collected as raw materials for polysaccharide extraction, and stored
at room temperature in a desiccator until use.
DEAE-Sepharose (fast flow) was purchased from GE Healthcare
Life Science (Shanghai, China). Fluorescein sodium salt, 2,20 -azo-
bis(2-methylpropionamidine)-dihydrochloride (AAPH), 6-hydroxy-
2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,20 -azino-
bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt
(ABTS), monosaccharide standards of rhamnose, arabinose, fucose,
xylose, mannose, glucose and galactose, and a uronic acid reference
were purchased from Sigma Chemical Company (St. Louis, MO,
USA). All other chemicals and solvents were of the highest com-
mercial grade and obtained from Sinopharm Chemical Reagent Co.
Ltd.
2.2. Preparation of polysaccharides
Prior to the citric acid extraction, the Laminaria japonica powder
was extracted by hot water (1: 30, w/v) at 120  C for 3 h to remove
water-soluble polysaccharide via centrifugation (3000g, 15 min).
Then the resultant supernatant was removed and the pellet resi-
dues were dried at 40  C for further citric acid extraction. Briefly,
100 g of the residues were treated with 3 l pH 2.0 of citric acid for
3 h at 120  C. The extract was collected, neutralized and concen-
trated to about 600 ml using a rotary evaporator at 55  C. Anhy-
drous ethanol was then added to a final ethanol concentration of
80% (v/v), which was kept overnight at 4  C, and centrifuged at
4000g for 20 min to obtain citric acid extracted crude poly-
saccharides. The precipitate was deproteinated with Sevag reagent
(chloroform: n-butyl alcohol ¼ 4: 1, v/v), and was dialyzed and
lyophilized to obtain Laminaria japonica polysaccharides (LJPA-P)
with the yield of 7.56%.
2.3. Separation and purification of crude polysaccharide (LJPA-P)
The lyophilized LJPA-P was dissolved in distilled water and
loaded on a DEAE-Sepharose fast flow column (2.5  20 cm) which
was eluted successively with distilled water, 0.1, 0.2, 0.3, 0.4, 0.5 and
0.6 M NaCl at a flow rate of 1.0 ml/min (LJPA-P0, LJPA-P1,LJPA-P2,
LJPA-P3, LJPA-P4, LJPA-P5 and LJPA-P6 fractions corresponding to
the six NaCl elutions). Each fraction with 5 ml of eluate was
collected, dialyzed and lyophilized for further investigation.
603
2.4. Chemical composition determination
Total sugar content was determined for calculation of purity
using the phenol-sulphuric acid method using glucose as the
standard (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). Sulfate
content was analyzed using barium chloride-gelatin method
(Dodgson & Price, 1962). The acidic monosaccharide uronic acid
(estimated as galacturonic acid and glucuronic acid contents) were
analyzed by Dionex ion chromatography ICS 5000 (Sunnyvale, CA,
USA) using an IonPac AS23 separator column (4  250 mm, i.d.,
5 mm), and the neutral monosaccharide profile was analyzed by gas
chromatography based on the method described by Rostamia and
Gharibzahedib (2016).
2.5. Methylation analysis
Methylation analysis was carried out by the method of You et al.
(2013). Five milligrams of purified polysaccharide was precisely
weighed and dissolved in 5.0 ml of DMSO in a sonication water
bath. Then, 200 mg of NaOH was added before the mixture was
ultrasonic treated for 30 min. Methylation reaction started with the
addition of 2.5 ml methyl iodide. The methylated polysaccharides
were extracted with 5  4 ml of chloroform, and the chloroform
layer was washed with 5  4 ml of distilled water. Then, the
chloroform was dried at reduced pressure using a rotary evapo-
rator. The residue was hydrolysed with 10 ml 2 M trifluoroacetic
acid at 100  C for 2 h in a sealed tube, and excess trifluoroacetic acid
was removed by evaporation under reduced pressure. The residue
was dissolved in methanol and evaporated to dryness three times.
The hydrolysate was reduced by 100 mg of NaBH4. After reduction,
0.1 ml of glacial acetic acid was added. The mixture was dried under
reduced pressure. The residue was dissolved in methanol and
evaporated to dryness three times. The residue was redissolved in
2 ml of methanol, and then was evaporated to dryness with ni-
trogen. The residue was then acetylated by 2 ml of acetic anhydride
and 2 ml of pyridine at 90  C for 30 min. Two milliliter of distilled
water was used to stop the reaction. The acetylated derivatives
were extracted with 4 ml of methylene chloride. The methylene
chloride layer was washed with 2 ml of distilled water, and treated
with anhydrous sodium sulfate. Finally, it was filtered through a
0.22 mm of nylon membrane for the gas chromatography/mass
spectrometer analysis.
2.6. Infrared spectrum analysis
The infrared spectra of LJPA-P, LJPA-P1, LJPA-P2 and LJPA-P3
were examined using a Fourier transform infrared (FT-IR) spec-
trophotometer (Bruker, Ettlingen, Germany) coupled with OPUS 3.1
software. The polysaccharide samples (~2e4 mg) were ground with
KBr powder and then pressed into pellets for FT-IR measurement
with a scanning range of 4000e500 cm1 (Abdelmalek et al., 2015).
2.7. Molecular weight determination
The molecular weights of purified polysaccharides were
measured using a Waters high-performance liquid chromatog-
raphy (Waters 1525, Waters Co. Ltd., Milford, MA, USA) equipped
with a Waters 2414 Refractive Index Detector. Separation was
performed with a TSK-GEL Guard Column (PWXL 6.0  40 mm), a
TSKGEL4000 K gel column (PWXL 7.8  300 mm) and a TSK-
GEL2500 K gel column (PWXL 7.8  300 mm) (TOSOH Co. Ltd.,
Tokyo, Japan) at temperature of 35  C. Phosphate buffer (0.2 M, pH
7.0) was used as the flow phase at a flow rate of 0.6 ml/min, and the
injection volume was 30 ml. The dextrans with different molecular
weights (4.4, 9.9, 21.4, 43.5, 124, 196, 277, 401, 1285 kDa) were used
604 C. Cui et al. / LWT - Food Science and Technology 73 (2016) 602e608

to obtain the calibration curve.

2.8. Determination of antioxidant activity

2.8.1. Oxygen radical absorbance capacity (ORAC) assay

ORAC was determined using a Varioskan Flash spectral scan
multimode plate reader (Thermo Fisher Scientific, Thermo Electron
Co. Waltham, MA, USA) with an excitation wavelength of 485 nm
and an emission filter at 538 nm according to the method of You
et al. (2013). The reaction was done in 75 mM phosphate buffer
(pH 7.4) with 200 ml of final reaction mixture. Fluorescein (40 ml,
3.5 nM) and 20 ml sample solution with different concentrations
were placed in the wells of the microplate preincubated for 15 min
at 37  C, and then rapidly added the AAPH solution (140 ml, 12 mM)
using a multichannel pipette. The microplate was immediately
placed in the reader and automatically shaken prior to each
reading. The fluorescence was recorded every 2 min for 120 min. A
blank with fluorescein and AAPH using the phosphate buffer
instead of the antioxidant solution and the Trolox as standard
compound were used with each microplate. The results were
expressed as Trolox equivalents (mmol Trolox/g), and is quantified
by integration of the area under the curve.

2.8.2. ABTS radical cation scavenging activity

An ABTS assay was performed according to the method of
Thetsrimuang, Khammuang, Chiablaem, Srisomsap, and Sarnthima
(2011). The ABTS radical cation was produced by reacting 7 mmol/l
ABTS stock solution with 2.45 mmol/l potassium persulfate (final
concentration) in dark at room temperature for 12e16 h. The ABTS
radical cation solution was diluted in distilled water to give an
initial absorbance (A734) of 0.7 before use. Twenty microliters of
samples (0.5e4.0 mg/ml) were added to 980 ml of ABTS radical
cation solution. The absorbance was measured at 734 nm by a
spectrophotometer after 30 min of incubation, and the results were
expressed as percentage inhibition of ABTS radical cation with a
blank control.
2.9. Statistical analyses
Data were expressed as mean ± standard deviation (SD) for at
least three replicates. One way analysis of variance was used to
determine significant difference at p < 0.05 using SPSS 11 (SPSS Inc.,
Chicago, USA).
3. Results and discussion
Fig. 1. DEAE-Sepharose fast flow chromatogram of the crude polysaccharides from
Laminaria japonica (LJPA-P) with distilled water, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 M NaCl
elutions.
exchanged with balance ions and had a great affinity to the chro-
matography column (Ye, Liu, Wang, Wang, & Zhang, 2012).
3.2. Chemical composition of polysaccharides
The monosaccharide composition, the sulfate group and uronic
acid contents of obtained polysaccharides were summarized in
Table 1. Six monosaccharides including rhamnose, fucose, xylose,
mannose, glucose and galactose were identified in the LJPA-P, LJPA-
P1 and LJPA-P2, which was consistent with previous report on
brown seaweeds (Ruperez, Ahrazem, & Leal, 2002), although LJPA-
P3 did not contain rhamnose, xylose, and glucose. Galactose was
the predominant monosaccharide in LJPA-P followed by glucose
Table 1
Monosaccharide composition, sulfate group content and glycosidic linkage of
extracted polysaccharide fractions.

3.1. Isolation and purification of polysaccharides Compositions LJPA-P LJPA-P1 LJPA-P2 LJPA-P3

Monosaccharide composition (molar ratio, %)

The crude polysaccharides LJPA-P were obtained from Laminaria Rhamnose 2.7 1.9 4.3 nd
japonica with a yield of 7.56% and with a purity of 67.37%. As shown Fucose 3.6 3.1 5.5 4.4
Xylose 2.7 7.6 6.0 nd
in Fig. 1, three independent elution peaks LJPA-P1, LJPA-P2 and
Mannose 10.5 22.3 24.8 3.7
LJPA-P3 with yields of 1.84%, 3.42% and 0.93%, and with purities of Glucose 15.8 36.4 4.9 nd
70.42%, 61.65% and 54.94%, derived from 0.1, 0.2 and 0.3 M NaCl Galactose 64.7 28.7 54.5 91.9
elutions, respectively, were obtained after purification of crude Sulfate group content (%) 4.0 nda nd 12.7
polysaccharides LJPA-P using a DEAE-Sepharose fast flow column. Uronic acid content (%)
Glucuronic acid 3.6 3.4 4.7 4.1
There might be an approximately 18% loss of polysaccharides due to Galacturonic acid nd nd 0.2 0.1
adsorption to the resin. Moreover, there was no obvious peak in the Molar ratio of glycosidic linkage (molar ratio, %)
water eluate due to that all the water-soluble polysaccharides had Galp-(1/ 7.2 6.8 7.2 6.6
been removed prior to the preparation of LJPA-P. The eluting order Glcp-(1/ 2.1 14.6 1.3 nd
/3)-Galp-(1/ 15.2 5.4 3.7 19.3
of LJPA-P1, LJPA-P2, and LJPA-P3 fractions which corresponds to the
/3)-Fucp-(1/ 1.8 1.9 1.5 1.7
increasing NaCl concentration indicated the role of electrostatic /3)-Rhap-(1/ 1.0 1.0 1.0 nd
interactions and the effect of increasing ionic strength. The ob- /6)-Galp-(1/ 2.4 3.5 2.4 nd
tained LJPA-P1, LJPA-P2 and LJPA-P3 fractions belonged to acidic /6)-Glcp-(1/ 1.8 4.1 nd nd
polysaccharides on the basis of the nature of DEAE as an anion /3,6)-Manp-(1/ 4.1 12.6 6.3 1
a
exchanger and the fact that negatively charged polysaccharides nd: Not detectable.
 C. Cui et al. / LWT - Food Science and Technology 73 (2016) 602e608 605

and mannose, suggesting the possible presence of galactan and involved in these studies (You et al., 2013). Moreover, great
mannan residues. The different distributions of these three methylation tended to reduce the polarity of polysaccharide due to
monosaccharides in LJPA-P1, LJPA-P2 and LJPA-P3 suggested that shielding effect and steric hindrance.
these sugar residues played an important role in the polarity and
affinity of polysaccharides. An exclusively high proportion (91.85%)
of galactose in LJPA-P3 also suggested its dominance as the back- 3.4. Infrared spectrum analysis of polysaccharides
bone units of LJPA-P3. Such a backbone was previously found in
Bryopsis plumose and exhibited good antioxidative effects (Song, The IR spectra of LJPA-P, LJPA-P1, LJPA-P2 and LJPA-P3 are
Zhang, Zhang, & Wang, 2010). Moreover, the molar ratio of galac- shown in Fig. 2. Signals at 3405e3423 and 1077 cm1 repre-
tose, fucose and mannose in LJPA-P3 (26.1: 1.3: 1) was different sented the stretching vibration angular vibration of OeH linkage
from that (9: 2: 2) of polysaccharide extracted from the same type which indicated strong inter- and intra-molecular interactions of
of seaweed i.e. Laminaria japonica (Xue et al., 2001). This might be the polysaccharide chains. Weak signals around
due to different extraction process, analysis methods and initial 2930e2933 cm1 resulted from CeH stretching vibrations. The
Laminaria japonica materials used in both studies (Ruperez et al., presence of uronic acids in the LJPA-P fractions was evidenced by
2002). In the present study, removal of cations by the chelating two characteristic signal bands around 1610 and 1420 cm1
effects citric acid, the self hydrolysis of polymer chain, and the
subsequent release of more monosaccharides occurred in the hot
and acidic extraction would result in the different monosaccharide
molar ratio.
Sulfate group was only found in LJPA-P and LJPA-P3, and the
sulfate group content in LJPA-P3 was 3 times as much as that in
LJPA-P. The uronic acid of the LJPA-P fractions in this study
occurred in the forms of glucuronic acid (major form) and/or
galacturonic acid, was found in all the LJPA-P fractions with LJPA-
P2 and LJPA-P3 being significantly higher than LJPA-P1 and LJPA-P,
which was in agreement with those found in alginic acid
nchez-Machado, Lo
(Sa pez-Cervantes, Lopez-Hernandez, Paseiro-
Losada, & Simal-Lozano, 2004). It should be noted that the sul-
fate groups and uronic acid enabled LJPA-P and its fractions
negatively charged, and chemically and biologically active, which
was important to the functional properties of polysaccharides
(Peng et al., 2012; Sun-Waterhouse, Melton, O’Connor, Kilmartin,
& Smith, 2008).

3.3. The glycosidic linkages of polysaccharides

Eight types of glycosidic linkages (associated with hexose resi-

dues) were detected in the LJPA-P fractions, whilst xylose linkage
(pentose-type) was absent (Table 1). LJPA-P, LJPA-P1 and LJPA-P2
consisted of similar methylated fragments although their molar
ratios differed. The molar ratio of /3)-Galp-(1/, /3,6)-Manp-
(1/ and Glcp-(1/ residues as 15.2: 4.1: 2.1, indicating LJPA-P
mainly contained galactopyranose, mannopyranose and glucopyr-
anose residues, especially galactopyranose (i.e. 1-linked, 1,3-linked
and 1,6-linked galactose in molar ratio of 7.2: 15.2: 2.4). The LJPA-P1
contained high proportion of glucans, galactans and mannans, with
backbone having Glcp-(1/, /6)-Glcp-(1/, Galp-(1/, /3)-Galp-
(1/, /6)-Galp-(1/ and /3,6)-Manp-(1/ in the molar ratio of
14.6: 4.1: 6.8: 5.4: 3.5: 12.6. LJPA-P2 contained galactose and
mannose residues with Galp-(1/, /3)-Galp-(1/, /6)-Galp-
(1/ and /3,6)-Manp-(1/ in a molar ratio of 7.2: 3.7: 2.4: 6.3.
Moreover, The presence of /3,6)-Manp-(1/ (i.e. 25.3% and 26.9%,
respectively for LJPA-P1 and LJPA-P2) indicated the possibility of
branching. In comparison, the glycosidic bonding of LJPA-P3
appeared simpler. Galactose residue dominated in LJPA-P3 i.e. a
Galp backbone with most /3)-Galp-(1/ and Galp-(1/ in a molar
ratio of 19.3: 6.6. Some /3)-Fucp-(1/ and /3,6)-Manp-(1/ but
rhamnose and glucose residues occurred in LJPA-P3. While the
molar ratio of glycosidic linkages in LJPA-P3 agreed with its
monosaccharide composition, there existed discrepancies between
this study and previous reports. Wang, Zhang, Zhang, Zhang, and
Niu (2010) claimed/3)-Fucp-(1/ linkage to the main chain, and
Peng et al. (2012) reported the (1 / 4)-glycosidic linked backbone.
All these observed discrepancies could be explained by the
different extraction process and Laminaria japonica materials Fig. 2. FT-IR spectra of LJPA-P, LJPA-P1, LJPA-P2 and LJPA-P3.
606 C. Cui et al. / LWT - Food Science and Technology 73 (2016) 602e608

Fig. 3. Molecular weights of LJPA-P1, LJPA-P2 and LJPA-P3.

corresponding to asymmetrical (C]O) and symmetrical (CeO)
stretching vibration of carboxylate group (Manrique & Lajolo,
2002; Sivam, Sun-Waterhouse, Perera, & Waterhouse, 2012).
The signals at 1414e1420 cm1 might also come from the
asymmetrical bending vibration of CH3. The weak signals around
1298e1302 cm1 corresponding to CeH bending (Galat, 1980)
were detected in LJPA-P, LJPA-P1 and LJPA-P2 but missing in LJPA-
P3. A band around 1235e1257 cm1 corresponding to the
asymmetric S]O stretching vibration was detected in both LJPA-
P and LJPA-P3, but missing in the other two fractions, suggesting
the presence of sulfate group. This result was consistent with the
compositional data of Table 1. The distinct peaks at
1034 ± 1 cm1 in the spectra of the four fractions indicated the
presence of CeC stretching and/or alduronic acid, which possibly
due to symmetric CeO vibration associated with a CeOeSO3
group (Lucassen, Van Veen, & Jansen, 1998). The signal at
962 cm1 derived from the asymmetrical stretching vibration of
CeOeS bonds appeared in the spectra of LJPA-P2 (detected and
labelled), LJPA-P and LJPA-P1 (both detected but weaker) but
disappeared in the spectrum of LJPA-P3. This suggested the
linkage of the sulfated group in LJPA-P3 could be different from
others. The signal peaks around 889e892 cm1 suggested the
presence of b-linked pyranose residue (Coimbra, Goncalves,
Barros, & Delgadillo, 2002) and its intensity appeared to
decrease in the order of LJPA-P > LJPA-P2 > LJPA-P1 > LJPA-P3.
Signals around 816e819 cm1 were assigned to CeOeS stretch-
ing vibration of sulfate groups on galactopyranose residues.
 C. Cui et al. / LWT - Food Science and Technology 73 (2016) 602e608
3.5. Molecular weights of polysaccharides
There were different molecular weights for LJPA-P1, LJPA-P2 and
LJPA-P3 (Fig. 3). Based on the calibration curve of dextrans, the
average molecular weight of each fraction was estimated. LJPA-P1
and LJPA-P2 had only one peak with average molecular weight
being 19.07 and 30.34 kDa, respectively. LJPA-P3 had two peaks
with average molecular weight of 64.82 and 19.21 kDa. Such dif-
ferences in molecular weight suggested the differences in physical
properties including rheological behaviours, and a higher molecu-
lar weight tended to exhibit greater viscosity.
3.6. Antioxidant capacity of polysaccharides
3.6.1. Oxygen radical absorbance capacity (ORAC) assay
As shown in Fig. 4, the LJPA-P and its three derived fractions
exhibited different ORAC antioxidant activity. LJPA-P3 possessed
the highest antioxidant capacity (1247.22 mmol Trolox/g), which
was >3.5 and > 8.4 times as much as that of LJPA-P (305.77 mmol
Trolox/g), LJPA-P1 (129.01 mmol Trolox/g) and LJPA-P2 (135.53 mmol
Trolox/g), respectively. The high content of neutral monosaccharide
seemed to have negative impact on total antioxidant activity, whilst
the high acidic monosaccharide content such as uronic acid would
enhance total antioxidant activity (Sun-Waterhouse et al., 2008).
The sulfate group content might play a significant role in antioxi-
dant activity of polysaccharides due to that the sulfate group con-
tent in LJPA-P3 was the highest and 2 times higher than that of
LJPA-P (Table 1), which was consistent with that reported by
Song et al. (2010).
3.6.2. ABTS radical cation scavenging activity
The ABTS radical cation scavenging activity of LJPA-P and its
three fractions all increased with an elevated polysaccharide con-
centration (Fig. 5). LJPA-P3 had remarkably higher antioxidant ac-
tivity and greater activity increment as a function of polysaccharide
concentration, compared with the other three polysaccharides
during the detected concentration range. LJPA-P ranked the second
highest in terms of ABTS activity, exhibiting significantly stronger
scavenging ability than LJPA-P1 and LJPA-P2 within the detected
concentrations, and a trend was consistent with that of ORAC value.
Fig. 4. Oxygen radical absorbance capacity (ORAC) of LJPA-P, LJPA-P1, LJPA-P2 and
LJPA-P3. Different letters indicate significant difference, p < 0.05.
607
Fig. 5. ABTS radical scavenging activity of LJPA-P, LJPA-P1, LJPA-P2 and LJPA-P3.
In general, there was minimal difference in ABTS radical cation
scavenging activity between LJPA-P1 and LJPA-P2.
3.6.3. Relationships between structural characteristics and
antioxidant activity
Both ORAC and ABTS assays can be tailored for lipophilic and
hydrophilic antioxidants. The ORAC assay was based on a hydrogen
atom transfer mechanism, rather than a single electron transfer
mechanism. The ABTS assay measures the total antioxidant power
of a single compound and complex mixtures of various poly-
saccharides. In the present study, sulfation and monosaccharide
compositions (e.g. galactose content) appeared to be the most
influencing factors for the ORAC and ABTS activities of poly-
saccharides (such as LJPA-P3) (Lu et al., 2013; Melo, Pereira, Foguel,
& Mourao, 2004; Qi et al., 2005). Further, the glycosidic linkages
could also be critical for antioxidant capacity of the polysaccharides
e.g. the main chain with /3)-Galp-(1/ of LJPA-P3 might enable
considerable antioxidant activity. Sun, Li, and Liu (2010) found that
polysaccharide with a backbone consisting of 1,3-linked gal-
actopyranosyl, 1,3-linked and 1,3,6-linked mannopyranosyl had
stronger hydroxyl radical scavenging activity. Thus, the findings of
Sinha, Bandyopadhyay, and Ghosh (2011) who presented that
in vitro antioxidant capacities of polysaccharide with 1,3-linked/
1,3,6-linked galactosyl extracted from Senna were comparable to
those of standard antioxidants, and correlated with our results. In
comparison, molecular weight seemed to be the least contributor
to both the ORAC and ABTS activities (e.g. LJPA-P1 and LJPA-P2).
Polysaccharides are more likely act as secondary antioxidants,
rather than primary antioxidants, in a manner similar to the pro-
tective effect of pectin on ascorbic acid antioxidant reported by
Sun-Waterhouse et al. (2008). The sulfate polysaccharides showed
strong antioxidant activity, possibly due to the fact that the sulfate
group behaved like an electrophile to facilitate the intramolecular
hydrogen abstraction. Thus, LJPA-P polysaccharide fractions ob-
tained in this study can be utilised as a natural secondary antioxi-
dants, especially given the food safety-related drawbacks of
synthetic antioxidants like BHT (Kahl & Kappus, 1993).
4. Conclusions
In the present study, three polysaccharide fractions with
different ionic strength, composition, sulfation, glycosidic linkages
and molecular weight distribution were generated from Laminaria
japonica through sequential hot water and citric acid extraction,
further purification by DEAE-Sepharose fast flow chromatography
and serial elutions with increasing concentrations of NaCl solu-
tions. It was found that a combination of factors affected the anti-
oxidant activity of polysaccharides, especially sulfation,
608 C. Cui et al. / LWT - Food Science and Technology 73 (2016) 602e608
monosaccharide composition (e.g. galactose content) and glyco-
sidic linkages. Although low molecular weight is favorable, mo-
lecular weights seemed less critical for both the ORAC and ABTS
activities. Among all the polysaccharides, LJPA-P3 corresponding to
0.3 M NaCl elution had high sulfate group and galactose content,
and was unique in its molar ratio of galactose, mannose and fucose
(26.1: 1.3: 1). These characteristics along with the dominant
glycosidic linkages of 1,3- and 1-linked galactose enabled LJPA-P3
to possess remarkably high ORAC and ABTS activities. Therefore,
LJPA-P3 has the potential to be commercialised as a natural sec-
ondary antioxidant for health product applications. Future work
may focus on the improvement of LJPA-P3 production yield and
investigation on potential biological activities.
Acknowledgements
The authors gratefully acknowledge the National High Tech-
nology Research and Development Program 863 (No.
2014AA093602), National Natural Science Foundation of China
(Nos. 31101222 and 31201416), the Science and Technology Plan-
ning Project of Guangdong Province (No. 2016A010105003) and the
State Scholarship Fund of China Scholarship Council (No.
201506155043) for their financial support.
References
Abdelmalek, B. E., Sila, A., Krichen, F., Karoud, W., Martinez-Alvarez, O., Ellouz-
Chaabouni, et al. (2015). Sulfated polysaccharides from Loligo vulgaris skin:
Potential biological activities and partial purification. International Journal of
Biological Macromolecules, 72, 1143e1151.
Choi, J., Kim, H. J., & Lee, J. W. (2011). Structural feature and antioxidant activity of
low molecular weight laminarin degraded by gamma irradiation. Food Chem-
istry, 129, 520e523.
Coimbra, M. A., Goncalves, F., Barros, A. S., & Delgadillo, I. (2002). FTIR spectroscopy
and chemometric analysis of white wine polysaccharide extracts. Journal of
Agricultural and Food Chemistry, 50, 3405e3411.
Delattre, C., Fenoradosoa, T. A., & Michaud, P. (2011). Galactans: An overview of their
most important sourcing and applications as natural polysaccharides. Brazilian
Archives of Biology and Technology, 54, 1075e1092.
Dodgson, K. S., & Price, R. G. (1962). A note on the determination of the ester sulfate
content of sulfated polysaccharides. Biochemical Journal, 84, 106e110.
Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric
method for determination of sugars and related substances. Analytical Chem-
istry, 28, 350e356.
Galat, A. (1980). Study of the Raman scattering and infrared absorption spectra of
branched polysaccharides. Acta Biochimica Polonica, 27, 135e142.
Kahl, R., & Kappus, H. (1993). Toxicology of the synthetic antioxidants BHA and BHT
in comparison with the natural antioxidant vitamin E. Zeitschrift für
Lebensmittel-Untersuchung und eForschung, 196, 329e338.
€ger, A. K., & Staerk, D. (2016). Edible seaweed as
Liu, B., Kongstad, K. T., Wiese, S., Ja
future functional food: Identification of a-glucosidase inhibitors by combined
use of high-resolution a-glucosidase inhibition profiling and
HPLCeHRMSeSPEeNMR. Food Chemistry, 203, 16e22.
Liu, H. M., Wang, F. Y., & Liu, Y. L. (2016). Hot-compressed water extraction of
polysaccharides from soy hulls. Food Chemistry, 202, 104e109.
Lucassen, G. W., Van Veen, G. N. A., & Jansen, J. A. J. (1998). Band analysis of hydrated
human skin stratum corneum attenuated total reflectance fourier transform
infrared spectra in vivo. Journal of Biomedical Optics, 3, 267e280.
Lu, J., You, L., Lin, Z., Zhao, M., & Cui, C. (2013). The antioxidant capacity of
polysaccharide from Laminaria japonica by citric acid extraction. International
Journal of Food Science and Technology, 48, 1352e1358.
Manrique, G. D., & Lajolo, F. M. (2002). FT-IR spectroscopy as a tool for measuring
degree of methyl esterification in pectins isolated from ripening papaya fruit.
Postharvest Biology and Technology, 25, 99e107.
Melo, F. R., Pereira, M. S., Foguel, D., & Mourao, P. A. (2004). Antithrombin-mediated
anticoagulant activity of sulfated polysaccharides. Journal of Biological Chemis-
try, 279, 20824e20835.
Peng, Z., Liu, M., Fang, Z., Wu, J., & Zhang, Q. (2012). Composition and cytotoxicity of
a novel polysaccharide from brown alga (Laminaria japonica). Carbohydrate
Polymers, 89, 1022e1026.
Qi, H., Zhang, Q., Zhao, T., Chen, R., Zhang, H., Niu, X., et al. (2005). Antioxidant
activity of different sulfate content derivatives of polysaccharide extracted from
Ulva pertusa (Chlorophyta) in vitro. International Journal of Biological Macro-
molecules, 37, 195e199.
Rostamia, H., & Gharibzahedib, S. M. T. (2016). Microwave-assisted extraction of
jujube polysaccharide: Optimization, purification and functional characteriza-
tion. Carbohydrate Polymers, 143, 100e107.
Ruperez, P., Ahrazem, O., & Leal, J. A. (2002). Potential antioxidant capacity of
sulfated polysaccharides from the edible marine brown seaweed Fucus ves-
iculosus. Journal of Agricultural and Food Chemistry, 50, 840e845.
nchez-Machado, D., Lo
Sa  pez-Cervantes, J., Lo pez-Hernandez, J., Paseiro-Losada, P., &
Simal-Lozano, J. (2004). Determination of the uronic acid composition of
seaweed dietary fibre by HPLC. Biomedical Chromatography, 18, 90e97.
Sinha, S., Bandyopadhyay, S. S., & Ghosh, D. (2011). Structural characteristics, fluo-
rescence quenching, and antioxidant activity of the arabinogalactan protein-
rich fraction from senna (Cassia angustifolia) leaves. Food Science and Biotech-
nology, 20, 1005e1011.
Sivam, A. S., Sun-Waterhouse, D., Perera, C. O., & Waterhouse, G. I. N. (2012).
Exploring the interactions between blackcurrant polyphenols, pectin and wheat
biopolymers in model breads; a FT-IR and HPLC investigation. Food Chemistry,
131, 802e810.
Song, H., Zhang, Q., Zhang, Z., & Wang, J. (2010). In vitro antioxidant activity of
polysaccharides extracted from Bryopsis plumosa. Carbohydrate Polymers, 80,
1057e1061.
Sun-Waterhouse, D., Melton, L. D., O’Connor, C. J., Kilmartin, P. A., & Smith, B. G.
(2008). Effect of apple cell walls and their extracts on the activity of dietary
antioxidants. Journal of Agricultural and Food Chemistry, 56, 289e295.
Sun, Y., Li, T., & Liu, J. (2010). Structural characterization and hydroxyl radicals
scavenging capacity of a polysaccharide from the fruiting bodies of Auricularia
polytricha. Carbohydrate Polymers, 80, 377e380.
Sun, L., Wang, C., Shi, Q., & Ma, C. (2009). Preparation of different molecular weight
polysaccharides from Porphyridium cruentum and their antioxidant activities.
International Journal of Biological Macromolecules, 45, 42e47.
Thetsrimuang, C., Khammuang, S., Chiablaem, K., Srisomsap, C., & Sarnthima, R.
(2011). Antioxidant properties and cytotoxicity of crude polysaccharides from
Lentinus polychrous Le v. Food Chemistry, 128, 634e639.
Wang, J., Zhang, Q., Zhang, Z., Zhang, H., & Niu, X. (2010). Structural studies on a
novel fucogalactan sulfate extracted from the brown seaweed Laminaria
japonica. International Journal of Biological Macromolecules, 47, 126e131.
Xue, C. H., Fang, Y., Lin, H., Chen, L., Li, Z. J., Deng, D., et al. (2001). Chemical char-
acters and antioxidative properties of sulfated polysaccharides from Laminaria
japonica. Journal of Applied Phycology, 13, 67e70.
Ye, S., Liu, F., Wang, J., Wang, H., & Zhang, M. (2012). Antioxidant activities of an
exopolysaccharide isolated and purified from marine Pseudomonas PF-6. Car-
bohydrate Polymers, 87, 764e770.
You, L., Gao, Q., Feng, M., Yang, B., Ren, J., Gu, L., et al. (2013). Structural charac-
terization of polysaccharides from Tricholoma matsutake and their antioxidant
and antitumor activities. Food Chemistry, 138, 2242e2249.
Zeid, A. H. A., Aboutabl, E. A., Sleem, A. A., & El-Rafie, H. M. (2014). Water soluble
polysaccharides extracted from Pterocladia capillacea and Dictyopteris mem-
branacea and their biological activities. Carbohydrate Polymers, 113, 62e66.
Zhang, H., & Row, K. H. (2015). Extraction and separation of polysaccharides from
Laminaria japonica by size-exclusion chromatography. Journal of Chromato-
graphic Science, 53, 498e502.