Eur Food Res Technol (2009) 230:101–109
DOI 10.1007/s00217-009-1147-4
ORIGINAL PAPER
Antioxidant activities in vitro of ethanol extract from brown
seaweed Sargassum pallidum
Hong Ye Æ Chunhong Zhou Æ Yi Sun Æ Xin Zhang Æ
Jun Liu Æ Qiuhui Hu Æ Xiaoxiong Zeng
Received: 25 March 2009 / Revised: 3 September 2009 / Accepted: 13 September 2009 / Published online: 25 September 2009
Ó Springer-Verlag 2009
Abstract The crude extract (CE) was obtained by
extracting the powder of Sargassum pallidum with a
solution of 70% ethanol. Then, CE dissolved in distilled
water was fractionated with chloroform (Cf), ethyl acetate
(EtOAc), and n-butanol (n-BuOH), respectively, affording
four fractions of Cf, EtOAc, n-BuOH and aqueous. First,
the contents of total polyphenols, vitamin C (VC) and
vitamin E (VE) in CE and its four fractions were deter-
mined. As results, the contents of total polyphenols in CE
and its fractions decreased in the following order: aqueous
fraction [ n-BuOH fraction [ EtOAc fraction [ CE [ Cf
fraction. The aqueous fraction had significantly higher VC
content (1.82%) compared with CE and fractions of Cf,
EtOAc, and n-BuOH (P \ 0.05). The contents of VE in CE
and its fractions were all in low level compared with the
total polyphenol content and VC content. Second, the
antioxidant activities in vitro of CE and its four fractions
were evaluated. Among all the fractions, EtOAc fraction
exhibited the highest total antioxidant activity (0.52 lmol
FeSO4 equivalent/mg extract), while fractions of EtOAc
and n-BuOH exhibited the highest 2,2-diphenyl-1-picryl-
hydrazyl (DPPH) radical scavenging activity and capacity
of chelating iron ions, respectively. In addition, a higher
content of total polyphenols (52.08 mg chlorogenic acid
equivalent/g extract) and reducing power (0.505 at A700)
for aqueous fraction were noticed. Finally, it was found
H. Ye
Y. Sun
X. Zhang
J. Liu
Q. Hu
X. Zeng (&)
College of Food Science and Technology,
Nanjing Agricultural University, 1 Weigang,
210095 Nanjing, Jiangsu, People’s Republic of China
e-mail: zengxx@njau.edu.cn
C. Zhou
Jiangsu Environmental Monitoring Center, 210036 Nanjing,
Jiangsu, People’s Republic of China
that the extracts of S. pallidum contained large amounts of
phlorotannin dimers and trimer based on the analytical
results of ultra high performance liquid chromatography–
mass spectroscopy. The results suggest that S. pallidum can
be a good source of natural antioxidant.
Keywords Antioxidant activity
Capacity of chelating
iron ions
DPPH
Polyphenol content

Sargassum pallidum
UHPLC–MS
Introduction
In the biomedical area, seaweeds have attracted an emerging
interest mainly for their bioactive substances, which have
great chances to be used as antioxidant, antimicrobial, anti-
viral, and anti-tumor drugs [1, 2]. More recently, it has been
demonstrated that seaweeds are rich source of antioxidants,
such as phylopheophytin in Eisenia bicycis, fucoxanthine in
Hijikia fusiformis and phlorotannins in Sargassum kjella-
manianum [3–7]. Furthermore, several researchers have
investigated the antioxidant properties of both brown and red
seaweeds [8, 9]. Sargassum pallidum, one kind of brown
seaweeds extensively distributed in Yellow Sea and East
China Sea, is rich in dihomogammalinolenic acid, amino
acids, polysaccharides, and trace elements [10]. In our pre-
vious study, we found that polysaccharides from S. pallidum
with relatively lower molecular weight (\50 kDa) had sig-
nificantly higher anti-tumor activity against the HepG2 cells,
A549 cells and MGC-803 cells in vitro. The IC50 value of
anti-tumor activity in vitro against the human gastric cancer
cell line MGC-803 was 0.125 mg/ml [11]. However, little
information about the content of phenolic compounds and
antioxidant activity of S. pallidum is available when com-
paring with other brown seaweeds [8, 12].
123
102
Phenolic compounds are ubiquitous bioactive com-
pounds and in the form of secondary metabolites univer-
sally existed in higher plants [13]. Accordingly, bioactive
polyphenols have attracted special attention because they
can protect the human body from the oxidative stress,
which may cause many diseases, including cancer, car-
diovascular dysfunction, and aging [14]. More recent
reports revealed seaweeds to be a rich source of phenolic
compounds. Furthermore, there is not any report about the
antioxidant activities and characterization of chemical
structures of the phenolic compounds in the brown alga S.
pallidum. Therefore, the objectives of the present study
were to evaluate the antioxidant activity in vitro and to
characterize the phenolic compounds in S. pallidum. First,
the crude extract (CE) was obtained by extracting the
powder of S. pallidum with 70% ethanol. CE dissolved in
distilled water was then fractionated with chloroform (Cf),
ethyl acetate (EtOAc) and n-butanol (n-BuOH), respec-
tively, affording four fractions of Cf, EtOAc, n-BuOH, and
aqueous. The contents of total polyphenols, vitamin C
(VC), and vitamin E (VE) in CE and its four fractions were
determined. Second, the antioxidant activities of CE and its
four fractions were investigated by using various in vitro
free-radical scavenging procedures. Finally, the main
phenolic compounds in CE and its four fractions were
analyzed by ultra high performance liquid chromatogra-
phy–mass spectroscopy (UHPLC–MS) with electrospray
ionization source (ESI).
Materials and methods
Materials and reagents
Sargassum pallidum was collected freshly (Weihai, Shan-
dong province, China), washed thoroughly with deionized
water and dried in shade at 20 °C. The dried seaweeds
were then powdered and stored at -20 °C until use.
Chlorogenic acid (CHA) and 2,2-diphenyl-1-picrylhy-
drazyl (DPPH) were obtained from Sigma Chemical Co.
(St. Louis, MO, USA). Folin–Ciocalteu reagent and 2,4,6-
tri(2-pyridyl)-s-triazine (TPTZ) were obtained from Fluka
(Tokyo, Japan). Assay kits for VC and VE were the prod-
ucts of Nanjing Jiancheng Bioengineering Institute (Nan-
jing, China). All other reagents were of analytical grade.
Preparation of extracts of S. pallidum
The powder of S. pallidum was macerated with 70% eth-
anol at a ratio of 1:10 (w/v) for 24 h under dark condition.
After centrifugation at 5000g for 10 min, the residue was
re-extracted twice with 70% ethanol as described above.
The supernatants were pooled together, evaporated by a
123
Eur Food Res Technol (2009) 230:101–109
rotary evaporator, and freeze-dried to afford CE. Then, CE
was dissolved in distilled water and fractionated through
solvent–solvent partitioning with Cf, EtOAc, and n-BuOH,
respectively. The resulting fractions including aqueous
were concentrated and freeze-dried, respectively. The CE
and its four fractions were stored in dark at 4 °C for use.
Determination of the contents of polyphenols, vitamin
C, and vitamin E
The total polyphenol contents of CE and its fractions were
measured by the Folin–Ciocalteu method described by
Nurmi with slight modification [15]. Briefly, an aliquot of
0.5 ml of sample solution (with appropriate dilution to
obtain absorbance in the range of the prepared calibration
curve) was mixed with 1.0 ml of Folin–Ciocalteu reagent
(10 times dilution) and allowed to react for 5 min at 30 °C
in the dark. Then 2.0 ml of saturated Na2CO3 solution was
added and the mixture was allowed to stand for 1 h before
the absorbance of the reaction mixture was read at 747 nm.
A calibration curve of CHA (ranging from 0.02 to 0.10 mg/
ml) was prepared, and the total polyphenol content was
standardized against CHA and was expressed as mg CHA
equivalents per gram of sample on a dry weight (DW)
basis.
The contents of VC and VE in CE and its fractions were
determined by using the assay kits for VC and VE according
to the instructions on the kits based on the method of Aly
[16] and the method of Adeniyi and Jaselskis [17],
respectively.
Total antioxidant activity
The abilities to reduce ferric ions of CE and its fractions
were measured using a modified version of the method
described by Benzie and Strain [18]. An aliquot (1.0 ml) of
each sample (with appropriate dilution if necessary) was
added to 5.0 ml of ferric ion reducing antioxidant power
(FRAP) reagent (10 parts of 0.3 mol/l sodium acetate
buffer at pH 3.7, 1 part of 0.01 mol/l TPTZ solution, and 1
part of 0.02 mol/l FeCl3
6H2O solution), and the reaction
mixture was incubated at 37 °C for 10 min. The increase in
absorbance at 593 nm was measured. Fresh working
solutions of FeSO4 were used for calibration. The antiox-
idant capacity based on the ability to reduce ferric ions was
calculated from the linear calibration curve and expressed
as mmol FeSO4 equivalents per gram of sample (DW).
DPPH radical scavenging activity
The DPPH free-radical scavenging activities of CE and its
fractions were determined according to the method
described by Leong et al. [19]. The initial absorbance of the
Eur Food Res Technol (2009) 230:101–109
DPPH in ethanol was measured at 517 nm and did not
change throughout the period of assay. An aliquot (0.1 ml)
of each sample (with different concentration) was added to
3.0 ml of ethanolic DPPH solution. Discolorations were
measured at 517 nm after incubation for 30 min at 30 °C in
dark. CHA was used as the positive control. The percent-
age of DPPH which was scavenged was calculated using
the following formula:

  30%; 18–20 min, A 70%, B 30%. The flow rate was set at
Scavenging % ¼ 1  Asample  Ablank Acontrol  100%
Here, ethanol (3.0 ml) plus sample solution (0.1 ml) was
used as a blank and 3 ml DPPH–ethanol solution plus
ethanol (0.1 ml) was used as a negative control.
Reducing power
Reducing powers of CE and its fractions were determined
by the method described by Oyaizu [20]. Briefly, an aliquot
(1.0 ml) of each sample (with different concentration) was
mixed with 1.0 ml of phosphate buffer (0.2 mol/l, pH 6.6)
and 1.0 ml potassium ferricyanide (1%). Reaction mixture
was incubated at 50 °C for 20 min. After incubation,
1.0 ml of trichloroacetic acid (10%) and 0.2 ml FeCl3
(0.1%) was added. The mixture was incubated for 10 min.
Absorbances of all the sample solutions were measured at
700 nm. VC was used as the positive control. Increased
absorbance indicates increased reducing power.
Capacity of chelating iron ions
The iron ion-chelating activity was determined by the
method of Dinis et al. [21]. Briefly, an aliquot (1.0 ml) of
each sample (with different concentration) was mixed with
0.05 ml FeCl2 (2.0 mmol/l), 0.2 ml ferrozine (5.0 mmol/l)
and 2.75 ml distilled water. The mixture was shaken vig-
orously at room temperature in the dark for 10 min, and the
absorbance of the iron ions–ferrozine complex at 562 nm
was measured. EDTA was used as the positive control. The
ability of sample to chelate iron ions was calculated using
the following equation:

  and n-BuOH, four fractions of CE were obtained. Among
Chelating activity ð%Þ ¼ 1  Asample  Ablank Acontrol
 100%
Here, FeCl2 solution substituted by distilled water was used
as a blank, and the sample substituted by distilled water
was used as a negative control.
UHPLC–MS analysis
The CE and its fractions were analyzed using a Waters
ACQUITY system (Waters Co., Ltd., USA) equipped with
a binary solvent manager, sample manager, and MS
103
spectrometer with an ESI interface and a ternary quadruple
mass analyzer. The separation was completed on a Bridged
Ethyl Hybrid (BEH) C18 columm (2.1 9 100 mm, 1.7 lm)
(Waters Co., Ltd., USA). The oven temperature was set at
25 °C. The mobile phase consisted of methanol (A) and
ultra-pure water (B). Elution was performed with a linear
gradient as follows: 0–10 min, A from 70 to 0%, B from 30
to 100%; 10–18 min, A from 0 to 70%, B from 100 to
0.3 ml/min. The ion source temperature and desolvation
temperature were maintained at 120 and 380 °C, respec-
tively. The capillary voltage and cone voltage were set at
2.5 and 25 V, respectively. Nitrogen was used as desolv-
ation gas at a flow rate of 500 L/h. Mass spectrum was
recorded in the range of m/z 100–2,000.
Statistical analysis
All the data were presented as mean ± standard deviations
of three determinations. Statistical analyses were per-
formed using student’s t test and one-way analysis of
variance. Multiple comparisons of means were done by the
least significance difference (LSD) test. All computations
were done by employing the statistical software (SAS,
version 8.0).
Results and discussion
Extraction yields of crude extract and its fractions
of S. pallidum
Due to the complicated constituents and pharmacological
diversities of seaweed materials, in vitro bioassay-guided
fractionation has been effectively applied to screen the
biological activities that provide important indications for
investigating the characteristics of active components [22].
In the present study, a solution of 70% ethanol was used to
extract S. pallidum, and a yield of 12.73% was obtained for
CE. Through solvent–solvent partitioning with Cf, EtOAc
the fractions, the highest yield was observed in Cf fraction
(42.90%), followed by aqueous fraction (28.08%), EtOAc
fraction (5.62%), and n-BuOH fraction (1.87%) as shown
in Table 1.
Phenolic compounds are commonly found in plants and
have been reported to have several biological activities
including antioxidant properties. In the present study, the
total phenolic contents of CE and its four fractions were
determined using Folin–Ciocalteu method. As results, the
total phenolic content of CE and its fractions decreased in
the following order (Table 1): aqueous fraction [ fraction
of n-BuOH [ fraction of EtOAc [ CE [ fraction of Cf.
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104
Table 1 Component analysis, total antioxidant capacity and yield of crude extract (as %w/w of S. pallidum on dry weight basis) and fractions
(as % of crude extract) of S. pallidum
Sample CE
Yield (%) 12.73 ± 0.02a
Total polyphenolcontent (mg CHA/g extract) 5.34 ± 0.02a
Vitamin C content (%) 0.48 ± 0.01a
Vitamin E content (%) 0.04 ± 0.01a
Total antioxidant status (9lmol FeSO4/mg) 0.14 ± 0.02a
Data are expressed as mean ± standard deviation
Values within a row followed by different letters are significantly different at P \ 0.05 using student’s t test
The aqueous fraction had significantly higher content of
phenolic compounds (52.08 mg CHA/g of extract) com-
pared with the other fractions and CE (P \ 0.05). The
lowest total phenolic content was found in fraction of Cf.
For VC contents of CE and its fractions, they decreased in
the following order: aqueous fraction [ CE [ fraction of
n-BuOH [ fraction of EtOAc [ fraction of Cf. It was
obvious that aqueous fraction had significantly higher
(P \ 0.05) VC content (1.82%) compared with CE and
fractions of Cf, EtOAc, and n-BuOH (P \ 0.05). For the
contents of VE in CE and its fractions, they were all in low
level when compared with total polyphenol content and VC
content, indicating less VE in the extract of S. pallidum.
Total antioxidant activity
FRAP assay is based on the ability of antioxidant to reduce
Fe3? to Fe2? in the presence of TPTZ, forming an intense
blue Fe2?-TPTZ complex with an absorption maximum at
593 nm. This reaction is pH-dependent. The absorbance
increase is proportional to the antioxidant content. Being
expressed in FeSO4 equivalents, the FRAP value was
applied to determine the antioxidant abilities of CE and its
fractions of S. pallidum. As results, total antioxidant
activity of CE and its fractions decreased in the following
order: EtOAc fraction [ n-BuOH fraction [ CE = Cf
fraction [ aqueous fraction. The highest activity of
0.52 lmol FeSO4/mg extract was observed in EtOAc
fraction, as compared with the other fractions and CE
(Table 1). Although aqueous fraction had relatively higher
content of polyphenols, it exhibited much lower total
antioxidant activity. As we know, hundreds of different
structures of phenolics are known in brown algae, most of
which are phlorotannins. In addition, the antioxidant
activities of phenolic compounds were different due to
their different structures. Therefore, there is a wide degree
of variation between different phenolic compounds in their
effectiveness as antioxidant. Although the aqueous fraction
had significantly higher (P \ 0.05) content of phenolic
compounds, these phenolic compounds showed lower
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Eur Food Res Technol (2009) 230:101–109
Cf fraction EtOAc fraction n-BuOH fraction Aqueous fraction
42.90 ± 0.02b 5.62 ± 0.01c 1.87 ± 0.01d 28.08 ± 0.02e
1.80 ± 0.01b 11.54 ± 0.01c 12.12 ± 0.02c 52.08 ± 0.02d
0.06 ± 0.01b 0.07 ± 0.02b 0.19 ± 0.01c 1.82 ± 0.02d
0.08 ± 0.02a 0.03 ± 0.01a 0.01 ± 0.00a 0.01 ± 0.01a
0.14 ± 0.01a 0.52 ± 0.01b 0.35 ± 0.01c 0.06 ± 0.00d
antioxidant activities than those with different structures.
Maybe this is the reason that aqueous fraction exhibited
lower total antioxidant activity.
DPPH free-radical scavenging activity
The DPPH free-radical is a stable free-radical, which has
been widely accepted as a tool for estimating the free-
radical scavenging activity of antioxidant [23]. Free-radi-
cals are highly reactive species made of molecules or atoms
that are unstable owing to single or unbalanced electrons.
Although free-radicals at physiological concentration may
be required for normal cell function, excessive amount of
free-radicals can damage cellular components such as lip-
ids, protein, and DNA [24]. Thus, the free-radical scav-
enging activities of antioxidants can protect the human
body from serious cellular or molecular damage by free-
radicals and retard the progress of many chronic diseases,
as well as lipid peroxidation in food. There are some
reports in the literature on the antioxidant capacity of algae.
Alcoholic and aqueous extracts of seaweeds have been
evaluated for antioxidant activity by lipoxygenase inhibi-
tion, DPPH assay, and deoxyribose assays [25, 26]. In the
present study, we found that the DPPH free-radical scav-
enging activities of EtOAc fraction and n-BuOH fraction
were much higher than those of the other fractions and CE
(Table 2). At the concentration of 2.0 mg/ml, the scav-
enging activities for EtOAc fraction and n-BuOH fraction
were 30.50 and 29.36%, respectively. While, DPPH radical
scavenging activity of CHA (positive control) was 29.81%
at the concentration of 0.1 mg/ml. In addition, the scav-
enging effects increased with the increase of sample con-
centration in experimental scope.
Reducing power
The reducing powers of CE and its fractions increased with
the increase of concentrations. The same trend has also been
reported by Kumaran and Karunakaran for methanol extracts
of higher plants [13]. It was observed that the aqueous
Eur Food Res Technol (2009) 230:101–109 105

Table 2 Antioxidant activities of crude extract and its fractions as assessed by DPPH method
Sample Concentration (mg/ml)
0.125 0.25 0.5 1.0 2.0

CE 5.00 ± 0.00a 6.31 ± 0.00b 6.07 ± 0.01b 8.10 ± 0.01c 11.55 ± 0.00d
Cf fraction 3.21 ± 0.00a 3.33 ± 0.01a 3.40 ± 0.01a 4.11 ± 0.00b 4.32 ± 0.01b
EtOAc fraction 2.29 ± 0.01a 6.60 ± 0.00b 10.80 ± 0.01c 17.97 ± 0.00d 30.50 ± 0.03e
n-BuOH fraction 1.33 ± 0.00a 4.64 ± 0.00b 8.33 ± 0.01c 19.70 ± 0.01d 29.36 ± 0.02e
Aqueous fraction – 0.48 ± 0.02a 0.95 ± 0.00b 3.81 ± 0.00c 6.10 ± 0.01d
Data are expressed as mean ± standard deviation
Values within a row followed by different letters are significantly different at P \ 0.05 using student’s t test
– Not detected

fraction showed the highest reducing power in all the frac-
tions and CE at the highest concentration (Table 3). At the
concentration of 0.1 mg/ml, the reducing power for aqueous
fraction was 0.322, while it was 0.364 for VC (positive
control) at a concentration of 8 lg/ml. The result indicates
that aqueous fraction has a considerable reducing power.
Chelating activity on iron ions
The method depends on the ability of sample to chelate
transition metals. In addition to initiating lipid peroxida-
tion, which leads to the deterioration of food, metal ions
possessing catalytic ability have been correlated with
incidence of arthritis and cancer. Additionally, ferrous ions,
commonly found in the food system, are considered the
most effective pro-oxidants. Therefore, the chelating abil-
ity of the extract from S. pallidum was examined in the
present study. It is well known that ferrozine can quanti-
tatively form complexes with Fe2?. However, in the pres-
ence of chelating agents, the complex formation is limited
and further results in a decrease in the red color of the
complex. The chelating effects of CE and its fractions on
iron ions were shown in Table 4. Most fractions showed
chelating ability. Especially, EtOAc fraction and n-BuOH
fraction exhibited higher chelating abilities compared with
the other fractions. At a concentration of 2 mg/ml, the

Table 3 Reducing power of crude extract and its fractions

Sample Concentration (lg/ml)
12.5 25 50 100 200

CE 0.246 ± 0.006a 0.254 ± 0.009a 0.261 ± 0.009a 0.266 ± 0.017a 0.267 ± 0.001a
Cf fraction 0.344 ± 0.002a 0.349 ± 0.012a 0.365 ± 0.007a 0.370 ± 0.005a 0.377 ± 0.028a
EtOAc fraction 0.105 ± 0.000a 0.141 ± 0.005b 0.166 ± 0.000b 0.208 ± 0.002c 0.219 ± 0.002c
n-BuOH fraction 0.166 ± 0.001a 0.193 ± 0.005ab 0.223 ± 0.009b 0.246 ± 0.011b 0.265 ± 0.016b
Aqueous fraction 0.166 ± 0.010a 0.181 ± 0.008a 0.242 ± 0.001b 0.322 ± 0.011c 0.505 ± 0.009d
Data are expressed as mean ± standard deviation
Values within a row followed by different letters are significantly different at P \ 0.05 using student’s t test

Table 4 Capacity of chelating iron ions of crude extract and its fractions
Sample Concentration (mg/ml)
0.25 0.5 1.0 2.0

CE 9.91 ± 0.01a 15.46 ± 0.06b 21.62 ± 0.05c 33.32 ± 0.05d

Cf fraction 4.27 ± 0.00a 10.98 ± 0.05b 12.71 ± 0.03c 15.26 ± 0.07d
EtOAc fraction – 10.52 ± 0.01a 27.22 ± 0.01b 48.25 ± 0.03c
n-BuOH fraction 1.24 ± 0.00a 10.10 ± 0.01b 25.77 ± 0.02c 51.34 ± 0.02d
Aqueous fraction – – 5.12 ± 0.01a 17.21 ± 0.03b
Data are expressed as mean ± standard deviation
– Not detected
Values within a row followed by different letters are significantly different at P \ 0.05 using student’s t test

123
106 Eur Food Res Technol (2009) 230:101–109

chelating abilities were 48.25 and 51.34% for EtOAc CE CE SN:QBA135

14.94
11-Sep-2008 12:52:45

100

fraction and n-BuOH fraction, respectively. The chelating 3.73

ability of EDTA (positive control) was 42.81% at the 14.97

concentration of 0.04 mg/ml. In addition, the chelating 14.99

ability of test sample increased with the increase of con-

%
centration during 0.25 to 2.0 mg/ml. Therefore, the che-
3.17

lating activity of the extract from S. pallidum suggests that 14.92

it can serve as an antioxidant. 4.16 14.39

14.80

15.02

There are some reports in the literature on the antioxi- 1.42

1.96 2.49
4.20

4.46 5.93 6.07

6.68
6.96
7.79 8.27
8.63
10.23
11.15 11.48
15.69
15.74
19.22

dant capacity of Sargassum species. To date, the extracts of 0

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00
Time

S. ringgoldianum subsp. Coreanum, S. marginatum, S. CF

100
Cf fraction
3.71
SN:QBA135 11-Sep-2008 12:31:33

ringgoldianum, S. siliquastrum, S. polycystum, S. bovea-

num, and S. latiuscula have been demonstrated to have
strong antioxidant activities [8, 27–32]. The extract from S. 3.28 3.72

ringgoldianum subsp. coreanum showed high superoxide

%
anion radical-scavenging activity, about 120 lmol catechin 3.11
14.96

equivalent/ml [27]. The ethanol extract from S. ringgol-

dianum also showed strong scavenging activity against
superoxide anion radicals (1.0 lg/ml, IC50), which were
14.42

3.99 15.43
2.90 7.45 8.26 8.35 8.53

0 Time

approximately five times stronger than that of catechin. In 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

addition, the active fraction has been demonstrated to EA1 EtOAc fraction SN:QBA135 11-Sep-2008 11:49:09

3.53
100

contain a mixture of high-molecular weight polyphenols, 3.55

Detector response (%)

phlorotannins [28]. For S. marginatum, the EtOAc fraction 3.51

of crude extract exhibited high total antioxidant activity 3.16

(39.62 mg ascorbic acid equivalent/g extract) and DPPH 2.98

2.97
3.59
%

scavenging activity (23.16%, concentration of extract

used = 1,000 lg) [29]. At a concentration of 1.0 mg/mL,
the ethanol extract from S. siliquastrum showed a DPPH 3.95 14.92

radical scavenging capacity of 95.20% [8]. By using trolox

3.98
14.96
5.89 6.71 6.79 7.02 14.35
1.51 2.24 7.95 8.198.81

0 Time

equivalent antioxidant capacity (TEAC) and FRAP assays, 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

the methanolic extract of S. polycystum has been demon- BOH1 n-BuOH fraction SN:QBA135 11-Sep-2008 13:13:56

3.19
100
3.76

strated to have potential antioxidant activity [30]. The

3.79

water extract of S. boveanum exhibited noticeable scav- 2.40

3.16

enging effects (about 94% at a concentration of 3 mg/ml)

in DPPH free-radical scavenging assay [31]. In the present
%

study, EtOAc fraction exhibited the highest total antioxi-

1.42

14.95
14.97

dant activity (0.52 lmol FeSO4 equivalent/mg extract), and 14.99

the fractions of EtOAc and n-BuOH exhibited the highest 3.26

DPPH radical scavenging activity and capacity of chelating

6.71
3.29
1.68 11.94 14.80
2.66 7.21 7.73 8.03 8.37 8.94 10.26 10.85 11.84 12.10
4.12 4.63 5.40
0 Time
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

iron ions, respectively. In addition, a higher reducing

AQ SN:QBA135 11-Sep-2008 13:35:08

power for aqueous fraction was noticed. Therefore, all the 100
Aqueous fraction 14.98

results suggest that Sargassum species can be a good

14.97

source for natural antioxidants.

Phenolic constituents in extracts of S. pallidum

%

15.00

Since CE and its fractions of S. pallidum showed strong

antioxidant activities as mentioned above, the extracts were
3.27 3.88 14.82

analyzed by UHPLC–MS with a BEH-C18 column in order 1.26 8.97 9.22

2.45 2.64 7.92 15.72
7.84 11.55 11.79
0 Time
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

to characterize the phenol compounds in extracts of

Retention time (min)
S. pallidum. The mass spectra of the peaks in the ESI–MS
spectrum were obtained in positive ion mode, and the Fig. 1 UHPLC-Mass chromatograms of CE, Cf fraction, EtOAc
results are shown in Figs. 1 and 2. It was notably that the fraction, n-BuOH fraction, and aqueous fraction of S. pallidum

123
Eur Food Res Technol (2009) 230:101–109 107

Fig. 2 ESI-Mass spectra of (a)

peaks whose retention time at CE
0 8 0 9 1 1 0 0 7 3 6 0 (3 .1 6 9 ) S b (1 ,7 5 .0 0 )
2.40, 3.17, and 3.73 min with an 100
2 1 9 .0 5

[M–H]? ion at m/z 219 a, peaks 2 1 9 .2 5

1: Scan E S+
5 .2 1 e 6
whose retention time at 3.28 and 2 1 8 .9 2

4.16 min with an [M–H]? ion at

m/z 247 b and peak whose
retention time at 14.94 min with
an [M–H]? ion at m/z 353 c,
respectively

%
1 0 4 .6 8

4 1 5 .2 6

1 0 4 .8 8

1 9 6 .8 1
4 1 5 .6 6
1 0 5 .3 4
2 2 0 .2 4
1 1 3 .9 2 1 4 6 .3 9 1 9 6 .9 4
2 3 7 .2 6 4 1 6 .4 5
1 9 7 .3 4 4 4 5 .0 3
2 3 3 .2 4 2 5 5 .8 7 3 2 7 .1 5 3 3 8 .1 1 4 0 4 .9 0
2 6 9 .2 7 3 2 2 .3 3 3 8 1 .5 3 3 8 5 .3 6 4 3 3 .0 8 4 3 6 .8 5
0 m /z
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440

(b)
CE
0 8 0 9 1 1 0 0 7 4 7 2 (4 .1 5 5 ) S b (1 ,7 5 .0 0 )
2 4 7 .3 6
100

1: S can E S +
2 4 6 .9 6 9 .7 4 e 5

1 0 1 .0 5
Detector response (%)

2 6 5 .1 8
%

2 6 5 .3 1
1 0 1 .1 9

2 6 5 .0 5

1 0 4 .9 5 2 4 7 .7 6
4 1 5 .3 3

3 9 9 .0 3
2 2 8 .3 5 2 6 4 .5 2 3 9 7 .3 1 4 1 5 .0 6 4 4 5 .6 9 4 4 9 .5 2
0 m /z
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440

(c)
CE
080911007 1697 (14.937) Sb (1,72.00 )
100 353.22

1: Scan ES+
1.51e7
353.55
%

354.47

354.74

302.20 443.45 644.71

369.52
126.99 140.98 229.15 303.13 394.80 443.78 477.37 635.14
184.93 331.64 429.45 567.01
278.71 285.31
0 m/z
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640

m/z

phenolic constituents in extracts of S. pallidum were sep- the short-chained phlorotannins [33, 34]. In addition, the
arated well by reverse-phase chromatography with a short phenolic constituents in extracts of S. pallidum were found
BEH-C18 column. It confirms the results that reversed- to be phlorotannin dimers and trimers based on the MS data
phase chromatography can provide a better separation for and references [35, 36]. As shown in Fig. 2a, the peaks

123
108
with retention time at 2.40, 3.17, and 3.73 min all showed
an [M–H]? ion at m/z 219. They would be isomeric com-
pounds of phlorotannin dimers. The molecular formula was
deduced to be C12H10O4. The peaks with retention time at
3.28 and 4.16 min showed an [M–H]? ion at m/z 247 in the
ESI–MS spectrum (Fig. 2b), indicating a molecular for-
mula of C13H10O5. They were indicative of the derivatives
of phlorotannin dimers and might have a main structure as
phenyl-furan-phenyl with four hydroxyl groups and one
methyl group on the two phenyls. The peaks with retention
time at 14.94 min showed an [M–H]? ion at m/z 353 in the
ESI–MS spectrum (Fig. 2c). It had a molecular formula of
C19H12O7. It might be derivative of phlorotannin trimer,
having a structure as phenyl-dioxine-phenyl-furan-phenyl
with four hydroxyl groups and one methyl group on the
three phenyls. Phlorotannins, composed of phloroglucinol
units, are known as marine algal polyphenols and are found
in the form of organic polymers [35, 37]. Therefore, the
results are general in agree with those reports that
phlorotannins are the main phenolic compounds in brown
algae.
As shown in Fig. 1, most of the phlorotannin dimers
were found in EtOAc fraction and n-BuOH fraction. This
could be related to their higher total antioxidant capacity,
DPPH free-radical scavenging activity and capacity of
chelating iron ions as compared with the other fractions
and CE. For aqueous fraction, only phlorotannin trimer
was found. As mentioned above, aqueous fraction
showed much higher reducing power when compared
with the other fractions. However, its total antioxidant
capacity and DPPH free-radical scavenging activity were
quite weak. The results indicate that phlorotannin trimers
may be mainly related to the reducing power, while the
phlorotannin dimers may be mainly related to the total
antioxidant capacity, DPPH free-radical scavenging
activity and capacity of chelating iron ions. In addition,
both phlorotannin dimers and trimer were found in Cf
fraction. Although the concentration of Cf fraction was
much higher than the other fractions, they showed only
almost the same peak areas in the chromatogram as that
of the other fractions. Accordingly, it should contain
other main substances. The result was in coincidence
with the result of Table 1 that Cf fraction had the lowest
total phenolic content. Therefore, it is worth further
works on the characterization, antioxidant activity, and
structure–function relationship of phlorotannins in
S. palladium.
Conclusion
The total antioxidant activities, DPPH free-radical-scav-
enging activities, reducing powers and capacities of
123
Eur Food Res Technol (2009) 230:101–109
chelating iron ions of CE and its fractions of S. pallidum
were evaluated in vitro. As results, EtOAc fraction and n-
BuOH fraction exhibited higher antioxidant activities,
while the aqueous fraction showed the highest reducing
power in all the fractions and CE. The results indicate that
S. pallidum can be potential source of natural antioxidants.
In addition, large amounts of phlorotannin dimmers and
trimer were found in the extract of S. pallidum through
UHPLC–MS analysis. The antioxidant activity of S. pal-
lidum might be related to its phenolic substrates, phloro-
tannins. Therefore, the results provide useful information
of S. pallidum on pharmacological activities and potential
applications of such compounds as natural antioxidants in
different food/pharmaceutical products.
Acknowledgments This work was partly supported by a grant-in-
aid for scientific research from the National Natural Science Foun-
dation of China (No.30570415), a grant-in-aid for the Key Project
from Chinese Ministry of Education (No.108152), and a grant-in-aid
for Youth Innovation Fund from Nanjing Agricultural University
(No.KJ04015).
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