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

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

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 603

elucidated. 2.4. Chemical composition determination


Therefore, one objective of this study was to propose an
improved process for crude polysaccharide preparation from Total sugar content was determined for calculation of purity
Laminaria japonica by combining hot water extraction and citric using the phenol-sulphuric acid method using glucose as the
acid extraction. The second objective was to fractionate and purify standard (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). Sulfate
crude polysaccharide by DEAE-Sepharose fast flow chromatog- content was analyzed using barium chloride-gelatin method
raphy using water elution followed by increasing concentration of (Dodgson & Price, 1962). The acidic monosaccharide uronic acid
NaCl elutions, and then to identify their chemical structures by gas (estimated as galacturonic acid and glucuronic acid contents) were
chromatography/mass spectrometry and Fourier infrared spectro- analyzed by Dionex ion chromatography ICS 5000 (Sunnyvale, CA,
photometry. The last objective was to evaluate antioxidant activity USA) using an IonPac AS23 separator column (4  250 mm, i.d.,
of purified fractions of Laminaria japonica polysaccharide with ox- 5 mm), and the neutral monosaccharide profile was analyzed by gas
ygen radical absorbance capacity (ORAC) and ABTS radical scav- chromatography based on the method described by Rostamia and
enging activity, and to elucidate structure-activity relationship of Gharibzahedib (2016).
the polysaccharide fractions.
2.5. Methylation analysis
2. Materials and methods
Methylation analysis was carried out by the method of You et al.
2.1. Materials and chemicals (2013). Five milligrams of purified polysaccharide was precisely
weighed and dissolved in 5.0 ml of DMSO in a sonication water
Brown seaweed Laminaria japonica, collected on the coast of bath. Then, 200 mg of NaOH was added before the mixture was
Qingdao, Shandong, China in September 2014, was washed, sun- ultrasonic treated for 30 min. Methylation reaction started with the
dried and ground into a fine powder using a laboratory mill. The addition of 2.5 ml methyl iodide. The methylated polysaccharides
powder particles that passed through a 40-mesh sieve were were extracted with 5  4 ml of chloroform, and the chloroform
collected as raw materials for polysaccharide extraction, and stored layer was washed with 5  4 ml of distilled water. Then, the
at room temperature in a desiccator until use. chloroform was dried at reduced pressure using a rotary evapo-
DEAE-Sepharose (fast flow) was purchased from GE Healthcare rator. The residue was hydrolysed with 10 ml 2 M trifluoroacetic
Life Science (Shanghai, China). Fluorescein sodium salt, 2,20 -azo- acid at 100  C for 2 h in a sealed tube, and excess trifluoroacetic acid
bis(2-methylpropionamidine)-dihydrochloride (AAPH), 6-hydroxy- was removed by evaporation under reduced pressure. The residue
2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,20 -azino- was dissolved in methanol and evaporated to dryness three times.
bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt The hydrolysate was reduced by 100 mg of NaBH4. After reduction,
(ABTS), monosaccharide standards of rhamnose, arabinose, fucose, 0.1 ml of glacial acetic acid was added. The mixture was dried under
xylose, mannose, glucose and galactose, and a uronic acid reference reduced pressure. The residue was dissolved in methanol and
were purchased from Sigma Chemical Company (St. Louis, MO, evaporated to dryness three times. The residue was redissolved in
USA). All other chemicals and solvents were of the highest com- 2 ml of methanol, and then was evaporated to dryness with ni-
mercial grade and obtained from Sinopharm Chemical Reagent Co. trogen. The residue was then acetylated by 2 ml of acetic anhydride
Ltd. 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
2.2. Preparation of polysaccharides chloride layer was washed with 2 ml of distilled water, and treated
with anhydrous sodium sulfate. Finally, it was filtered through a
Prior to the citric acid extraction, the Laminaria japonica powder 0.22 mm of nylon membrane for the gas chromatography/mass
was extracted by hot water (1: 30, w/v) at 120  C for 3 h to remove spectrometer analysis.
water-soluble polysaccharide via centrifugation (3000g, 15 min).
Then the resultant supernatant was removed and the pellet resi- 2.6. Infrared spectrum analysis
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 The infrared spectra of LJPA-P, LJPA-P1, LJPA-P2 and LJPA-P3
3 h at 120  C. The extract was collected, neutralized and concen- were examined using a Fourier transform infrared (FT-IR) spec-
trated to about 600 ml using a rotary evaporator at 55  C. Anhy- trophotometer (Bruker, Ettlingen, Germany) coupled with OPUS 3.1
drous ethanol was then added to a final ethanol concentration of software. The polysaccharide samples (~2e4 mg) were ground with
80% (v/v), which was kept overnight at 4  C, and centrifuged at KBr powder and then pressed into pellets for FT-IR measurement
4000g for 20 min to obtain citric acid extracted crude poly- with a scanning range of 4000e500 cm1 (Abdelmalek et al., 2015).
saccharides. The precipitate was deproteinated with Sevag reagent
(chloroform: n-butyl alcohol ¼ 4: 1, v/v), and was dialyzed and 2.7. Molecular weight determination
lyophilized to obtain Laminaria japonica polysaccharides (LJPA-P)
with the yield of 7.56%. The molecular weights of purified polysaccharides were
measured using a Waters high-performance liquid chromatog-
2.3. Separation and purification of crude polysaccharide (LJPA-P) raphy (Waters 1525, Waters Co. Ltd., Milford, MA, USA) equipped
with a Waters 2414 Refractive Index Detector. Separation was
The lyophilized LJPA-P was dissolved in distilled water and performed with a TSK-GEL Guard Column (PWXL 6.0  40 mm), a
loaded on a DEAE-Sepharose fast flow column (2.5  20 cm) which TSKGEL4000 K gel column (PWXL 7.8  300 mm) and a TSK-
was eluted successively with distilled water, 0.1, 0.2, 0.3, 0.4, 0.5 and GEL2500 K gel column (PWXL 7.8  300 mm) (TOSOH Co. Ltd.,
0.6 M NaCl at a flow rate of 1.0 ml/min (LJPA-P0, LJPA-P1,LJPA-P2, Tokyo, Japan) at temperature of 35  C. Phosphate buffer (0.2 M, pH
LJPA-P3, LJPA-P4, LJPA-P5 and LJPA-P6 fractions corresponding to 7.0) was used as the flow phase at a flow rate of 0.6 ml/min, and the
the six NaCl elutions). Each fraction with 5 ml of eluate was injection volume was 30 ml. The dextrans with different molecular
collected, dialyzed and lyophilized for further investigation. 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 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
Thetsrimuang, Khammuang, Chiablaem, Srisomsap, and Sarnthima
elutions.
(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 exchanged with balance ions and had a great affinity to the chro-
radical cation solution was diluted in distilled water to give an matography column (Ye, Liu, Wang, Wang, & Zhang, 2012).
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 3.2. Chemical composition of polysaccharides
cation solution. The absorbance was measured at 734 nm by a
spectrophotometer after 30 min of incubation, and the results were The monosaccharide composition, the sulfate group and uronic
expressed as percentage inhibition of ABTS radical cation with a acid contents of obtained polysaccharides were summarized in
blank control. Table 1. Six monosaccharides including rhamnose, fucose, xylose,
mannose, glucose and galactose were identified in the LJPA-P, LJPA-
2.9. Statistical analyses P1 and LJPA-P2, which was consistent with previous report on
brown seaweeds (Ruperez, Ahrazem, & Leal, 2002), although LJPA-
Data were expressed as mean ± standard deviation (SD) for at P3 did not contain rhamnose, xylose, and glucose. Galactose was
least three replicates. One way analysis of variance was used to the predominant monosaccharide in LJPA-P followed by glucose
determine significant difference at p < 0.05 using SPSS 11 (SPSS Inc.,
Chicago, USA).
Table 1
3. Results and discussion 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) due to symmetric CeO vibration associated with a CeOeSO3
stretching vibration of carboxylate group (Manrique & Lajolo, group (Lucassen, Van Veen, & Jansen, 1998). The signal at
2002; Sivam, Sun-Waterhouse, Perera, & Waterhouse, 2012). 962 cm1 derived from the asymmetrical stretching vibration of
The signals at 1414e1420 cm1 might also come from the CeOeS bonds appeared in the spectra of LJPA-P2 (detected and
asymmetrical bending vibration of CH3. The weak signals around labelled), LJPA-P and LJPA-P1 (both detected but weaker) but
1298e1302 cm1 corresponding to CeH bending (Galat, 1980) disappeared in the spectrum of LJPA-P3. This suggested the
were detected in LJPA-P, LJPA-P1 and LJPA-P2 but missing in LJPA- linkage of the sulfated group in LJPA-P3 could be different from
P3. A band around 1235e1257 cm1 corresponding to the others. The signal peaks around 889e892 cm1 suggested the
asymmetric S]O stretching vibration was detected in both LJPA- presence of b-linked pyranose residue (Coimbra, Goncalves,
P and LJPA-P3, but missing in the other two fractions, suggesting Barros, & Delgadillo, 2002) and its intensity appeared to
the presence of sulfate group. This result was consistent with the decrease in the order of LJPA-P > LJPA-P2 > LJPA-P1 > LJPA-P3.
compositional data of Table 1. The distinct peaks at Signals around 816e819 cm1 were assigned to CeOeS stretch-
1034 ± 1 cm1 in the spectra of the four fractions indicated the ing vibration of sulfate groups on galactopyranose residues.
presence of CeC stretching and/or alduronic acid, which possibly
C. Cui et al. / LWT - Food Science and Technology 73 (2016) 602e608 607

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

Fig. 5. ABTS radical scavenging activity of LJPA-P, LJPA-P1, LJPA-P2 and LJPA-P3.
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 In general, there was minimal difference in ABTS radical cation
the highest antioxidant capacity (1247.22 mmol Trolox/g), which scavenging activity between LJPA-P1 and LJPA-P2.
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
3.6.3. Relationships between structural characteristics and
Trolox/g), respectively. The high content of neutral monosaccharide
antioxidant activity
seemed to have negative impact on total antioxidant activity, whilst
Both ORAC and ABTS assays can be tailored for lipophilic and
the high acidic monosaccharide content such as uronic acid would
hydrophilic antioxidants. The ORAC assay was based on a hydrogen
enhance total antioxidant activity (Sun-Waterhouse et al., 2008).
atom transfer mechanism, rather than a single electron transfer
The sulfate group content might play a significant role in antioxi-
mechanism. The ABTS assay measures the total antioxidant power
dant activity of polysaccharides due to that the sulfate group con-
of a single compound and complex mixtures of various poly-
tent in LJPA-P3 was the highest and 2 times higher than that of
saccharides. In the present study, sulfation and monosaccharide
LJPA-P (Table 1), which was consistent with that reported by
compositions (e.g. galactose content) appeared to be the most
Song et al. (2010).
influencing factors for the ORAC and ABTS activities of poly-
saccharides (such as LJPA-P3) (Lu et al., 2013; Melo, Pereira, Foguel,
3.6.2. ABTS radical cation scavenging activity & Mourao, 2004; Qi et al., 2005). Further, the glycosidic linkages
The ABTS radical cation scavenging activity of LJPA-P and its could also be critical for antioxidant capacity of the polysaccharides
three fractions all increased with an elevated polysaccharide con- e.g. the main chain with /3)-Galp-(1/ of LJPA-P3 might enable
centration (Fig. 5). LJPA-P3 had remarkably higher antioxidant ac- considerable antioxidant activity. Sun, Li, and Liu (2010) found that
tivity and greater activity increment as a function of polysaccharide polysaccharide with a backbone consisting of 1,3-linked gal-
concentration, compared with the other three polysaccharides actopyranosyl, 1,3-linked and 1,3,6-linked mannopyranosyl had
during the detected concentration range. LJPA-P ranked the second stronger hydroxyl radical scavenging activity. Thus, the findings of
highest in terms of ABTS activity, exhibiting significantly stronger Sinha, Bandyopadhyay, and Ghosh (2011) who presented that
scavenging ability than LJPA-P1 and LJPA-P2 within the detected in vitro antioxidant capacities of polysaccharide with 1,3-linked/
concentrations, and a trend was consistent with that of ORAC value. 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-
Fig. 4. Oxygen radical absorbance capacity (ORAC) of LJPA-P, LJPA-P1, LJPA-P2 and tions. It was found that a combination of factors affected the anti-
LJPA-P3. Different letters indicate significant difference, p < 0.05. 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- polysaccharide from Laminaria japonica by citric acid extraction. International
Journal of Food Science and Technology, 48, 1352e1358.
sidic linkages. Although low molecular weight is favorable, mo-
Manrique, G. D., & Lajolo, F. M. (2002). FT-IR spectroscopy as a tool for measuring
lecular weights seemed less critical for both the ORAC and ABTS degree of methyl esterification in pectins isolated from ripening papaya fruit.
activities. Among all the polysaccharides, LJPA-P3 corresponding to Postharvest Biology and Technology, 25, 99e107.
0.3 M NaCl elution had high sulfate group and galactose content, Melo, F. R., Pereira, M. S., Foguel, D., & Mourao, P. A. (2004). Antithrombin-mediated
anticoagulant activity of sulfated polysaccharides. Journal of Biological Chemis-
and was unique in its molar ratio of galactose, mannose and fucose try, 279, 20824e20835.
(26.1: 1.3: 1). These characteristics along with the dominant Peng, Z., Liu, M., Fang, Z., Wu, J., & Zhang, Q. (2012). Composition and cytotoxicity of
glycosidic linkages of 1,3- and 1-linked galactose enabled LJPA-P3 a novel polysaccharide from brown alga (Laminaria japonica). Carbohydrate
Polymers, 89, 1022e1026.
to possess remarkably high ORAC and ABTS activities. Therefore, Qi, H., Zhang, Q., Zhao, T., Chen, R., Zhang, H., Niu, X., et al. (2005). Antioxidant
LJPA-P3 has the potential to be commercialised as a natural sec- activity of different sulfate content derivatives of polysaccharide extracted from
ondary antioxidant for health product applications. Future work Ulva pertusa (Chlorophyta) in vitro. International Journal of Biological Macro-
molecules, 37, 195e199.
may focus on the improvement of LJPA-P3 production yield and Rostamia, H., & Gharibzahedib, S. M. T. (2016). Microwave-assisted extraction of
investigation on potential biological activities. jujube polysaccharide: Optimization, purification and functional characteriza-
tion. Carbohydrate Polymers, 143, 100e107.
Ruperez, P., Ahrazem, O., & Leal, J. A. (2002). Potential antioxidant capacity of
Acknowledgements sulfated polysaccharides from the edible marine brown seaweed Fucus ves-
iculosus. Journal of Agricultural and Food Chemistry, 50, 840e845.
The authors gratefully acknowledge the National High Tech- 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
nology Research and Development Program 863 (No.
seaweed dietary fibre by HPLC. Biomedical Chromatography, 18, 90e97.
2014AA093602), National Natural Science Foundation of China Sinha, S., Bandyopadhyay, S. S., & Ghosh, D. (2011). Structural characteristics, fluo-
(Nos. 31101222 and 31201416), the Science and Technology Plan- rescence quenching, and antioxidant activity of the arabinogalactan protein-
ning Project of Guangdong Province (No. 2016A010105003) and the rich fraction from senna (Cassia angustifolia) leaves. Food Science and Biotech-
nology, 20, 1005e1011.
State Scholarship Fund of China Scholarship Council (No. Sivam, A. S., Sun-Waterhouse, D., Perera, C. O., & Waterhouse, G. I. N. (2012).
201506155043) for their financial support. Exploring the interactions between blackcurrant polyphenols, pectin and wheat
biopolymers in model breads; a FT-IR and HPLC investigation. Food Chemistry,
131, 802e810.
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