J Appl Phycol (2008) 20:705–711
DOI 10.1007/s10811-007-9254-8

Antioxidant activities of phlorotannins isolated
from Japanese Laminariaceae
Toshiyuki Shibata & Kanji Ishimaru &Shigeo Kawaguchi & Hiromichi Yoshikawa &Yoichiro Hama

Received: 23 April 2007 / Revised and Accepted: 5 September 2007 / Published online: 6 October 2007
# Springer Science + Business Media B.V. 2007

Abstract The antioxidant activities of brown algal phlorotannins were evaluated using the inhibition of phospholipid
peroxidation in the liposome system, and by determining
radical scavenging activities against the superoxide anion
and 1,1-diphenyl-2-picrylhydrazyl (DPPH). Oligomers ofphloroglucinol (1,3,5-trihydroxybenzene), eckol (a trimer),
phlorofucofuroeckol A (a pentamer), dieckol and 8,8′-

T. Shibata (*)
Laboratory of Quality in Marine Products,
Department of Life Sciences,
Graduate School of Bioresources, Mie University,
1577 Kurimamachiya,
Tsu 514-8507, Japane-mail: shibata@bio.mie-u.ac.jp
K. Ishimaru
Department of Applied Biological Sciences,
Faculty of Agriculture, Saga University,
1Honjo,Saga 840-8502, JapanS. Kawaguchi
Department of Animal and Marine Bioresource Sciences,
Faculty of Agriculture, Kyushu University,
6-10-1 Hakozaki, Higashi-ku,
Fukuoka 812-8581, Japan
H. Yoshikawa
Department of Life, Environment and Materials Sciences,
Faculty of Engineering, Fukuoka Institute of Technology,
3-30-1 Wajiro-Higashi, Higashi-ku,
Fukuoka 811-0295, Japan
Y. Hama
Department of Applied Biochemistry and Food Sciences,
Faculty of Agriculture, Saga University,
1 Honjo,
Saga 840-8502, Japanbieckol (hexamers), isolated from the Laminarian brownalgae Eisenia bicyclis, Ecklonia cava and Ecklonia kurome,
showed potent inhibition of phospholipid peroxidation at
1 μM in the liposome system. The phlorotannins had
significant radical scavenging activities against the superoxide anion (50% effective concentration values: 6.5–
8.4 μM) and DPPH (50% effective concentration values:
12–26 μM), and were more effective compared to ascorbic
acid and α-tocopherol. For the purpose of using phlorotannins as functional food ingredients, the antioxidant
activity of a complex of crude phlorotannins and soybean
protein was examined. The complex had a pronounced
DPPH-radical scavenging activity. These results suggestthat phlorotannins are potent anti-inflammatory substances,and that the Laminariaceous brown algae, which areabundant in phlorotannins, may be useful as a new
functional foodstuff or supplement with anti-inflammatory
activity.
Keywords Brown algae . DPPH-radical . Liposome .
Phlorotannin–soy bean protein complex . Phaeophyta .
Polyphenols . Superoxide anion radical

Introduction
It is well-known that the oxidative damage to cell
membranes, DNA and/or proteins induced by free radicals
and reactive oxygen species may play a causative role inageing and lifestyle diseases. Therefore, it is believed that
inhibiting such damage by taking antioxidant supplements
could be an effective therapy for such diseases. Recent
epidemiological and clinical studies have indicated that
consumption of plant foods and drinks (e.g., tea, red wineand soybean products) moderate the risk of lifestyle diseases

706

J Appl Phycol (2008) 20:705–711(Messina et al. 1994; Sasazuki et al. 2000; Wu et al. 2001).
The beneficial effects of such foods and drinks are thought tobe due to the presence of polyphenols with antioxidant
activity, and many active substances (e.g., catechins, resveratrol and isoflavones) have been isolated (Jang et al. 1997;
Esaki et al. 1999a, b; Zhao et al 2001; Havsteen 2002;
Aggarwal and Shishodia 2006). Polyphenols—common
secondary metabolites—are found universally in plants(Haslam 1989; Herbert 1989). However, in stark contrast topolyphenols in terrestrial plants, the literature on theproperties of marine algal polyphenols from a humanphysiological viewpoint is very sparse. Marine algal polyphenols—phlorotannins—which have been found to exist
only within brown algae, are formed by the polymerizationof phloroglucinol (Ragan and Glombitza 1986; Nakamura etal. 1996). Previously, we isolated the phlorotannins eckol (aphloroglucinol trimer), phlorofucofuroeckol A (a pentamer),
dieckol and 8,8′-bieckol (hexamers) from the JapaneseLaminariaceous brown algae Eisenia bicyclis (Kjellman)
Setchell, Ecklonia cava Kjellman and Ecklonia kurome
Okamura, and reported their distribution (Shibata et al.
2004) and physiological properties (Shibata et al. 2002a, b,
2003). However, the effectiveness of phlorotannins against
free radicals is still obscure.
The aim of this study was to evaluate the antioxidant andfree radical scavenging activities of phlorotannins isolated
from Laminariaceous brown algae. As a first step, theactivities of the phlorotannins were determined using theinhibition of phospholipid peroxidation in the liposome
system, and their scavenging activities against the superoxide anion and 1,1-diphenyl-2-picrylhydrazyl (DPPH)
radicals. Thereafter, a phlorotannin–soybean protein complex was prepared and its function as an antioxidant
foodstuff assessed.

50% H2SO4

Materials and methods
The brown algae Eisenia bicyclis, Ecklonia cava andEcklonia kurome were collected from along the coasts of
the Itoshima Peninsula (33°37′N, 130°10′E) in Fukuoka
Prefecture and the Miura peninsula (35°08′N, 130°36′E) in
Kanagawa prefecture, Japan. The algae were washed withfiltrated seawater, air-dried and pulverized, and the algal
powders were then stored at −40°C until use.
Catechin and epigallocatechin gallate (EGCG) werekindly donated by Kurita Water Industries (Tokyo, Japan).
Extraction of phlorotannins from algal powders was
carried out according to the modified method of Folch etal. (1957) described in previous reports (Nagayama et al.
2002, 2003; Shibata et al. 2006b). Each of the phlorotannins in the crude extracts was purified on a column(1.5 cm i.d.×150 cm) of Wakogel C-300HG (Wako,
Tokyo, Japan) with CHCl 3 :methanol (MeOH):water(80:20:2, v/v) as the eluent (Shibata et al. 2002b, 2003).
The purity of the phlorotannins was checked by thin-layer
chromatography (TLC) and reversed-phase three-dimensional high-performance liquid chromatography (RP-3DHPLC). TLC plates (Silica Gel 60 F254, 0.25 mm; Merck,
Darmstadt, Germany), which had been activated at 120°C
for 5 min before use, were developed with CHCl3:MeOH:
water:acetic acid (65:25:4:3, v/v) (Shibata et al. 2002a,
2004, 2006b). Fifty percent H2SO4 (Krebs et al. 1969),
vanillin-H2SO4 (Krebs et al. 1969), and paprika pigment(Nakamura et al. 1991), were used as the detecting agentsfor organic compounds, for phenolic compounds, and forantioxidant substances, respectively. The HPLC system
consisted of a Waters 600 pump (Waters, Milford, MA), a
MCPD-3600 UV detector (Otsuka Electronics, Osaka,
Japan) and an Inertsil ODS-3 column (4.6 mm i.d.×

Vanillin -H2SO4

Paprika pigment
Front

P-1
P-2
P-3
P-4
P-5
P-6

a

b

c

d

a

b

Fig. 1 Thin-layer chromatography (TLC) of phlorotannins from theLaminariaceous brown algae Eisenia bicyclis, Ecklonia cava andEcklonia kurome. P-1 Phloroglucinol, P-2 phloroglucinol tetramer,
P-3 eckol, P-4 phlorofucofuroeckol A, P-5 dieckol, P-6 8,8′-

c

d

a

b

c

d

Originbieckol. a Phloroglucinol, b crude phlorotannins from Eisenia
bicyclis, c crude phlorotannins from Ecklonia cava, d crude
phlorotannins from Ecklonia kurome

J Appl Phycol (2008) 20:705–711

707

250 mm, GL Science, Tokyo, Japan). Elution was
performed at a flow-rate of 1.0 mL min−1 with a linear
gradient from 30% MeOH/water (eluent A) to 100%
MeOH (eluent B) for 20 min, and was maintained for 25
min (Shibata et al. 2002a, b, 2003, 2004, 2006b). The
photodiode array detector was set to a wavelength range of
190–400 nm.

A phlorotannin–soybean protein complex was prepared by
the method of Ishimaru and Nonaka (2001). Briefly,
defatted soybean (200 g) was steeped in water (1 L) andAntioxidant activity in the liposome systemaAbsorbanceP-3 P-5

P-4

P-1

P-2

0
200

300
Wavelength (nm)
400
0

b
1.5

Radical scavenging activities against DPPH and superoxide
anion

10
20
Retention time (min)

30

P-5
P-6

P-1

P-3

P-4

0
200

300
Wavelength (nm)4000cAbsorbance

The DPPH-radical scavenging activity was measured using
the modified method of Yamaguchi et al. (1998) describedin a previous report (Shibata et al. 2006a). The samples
were dissolved in 100 μL EtOH, and the solution was
mixed with 100 μL 300 mM DPPH (Wako) in EtOH. In
controls, 100 μL EtOH was used instead of the sample
solution. After the reaction mixture had been stored at room
temperature for 30 min, its absorbance was measured at
520 nm. The superoxide anion radical scavenging activity
was determined with the SOD assay kit-WST (Dojindo
Laboratories, Kumamoto, Japan) (Ukeda et al. 1999).
Samples were dissolved in 20 μL EtOH. In controls, 20 μL
EtOH was used instead of the sample solution. The
production of water-soluble tetrazolium salt was comparedwith that of controls.
The 50% effective concentration (EC50) value wasdefined as the amount of sample required to scavenge
50% DPPH radical or superoxide anion radical. Terrestrial
polyphenols (resveratrol, catechin and EGCG) and antioxidant vitamins (ascorbic acid and α-tocopherol) were usedas positive controls.

P-6

1.5

AbsorbanceA large unilamellar vesicle was prepared by the extrusion
method described by MacDonald et al. (1991) with somemodifications. Briefly, egg yolk lecithin (Wako, Tokyo,
Japan) in chloroform (10 mg mL–1) was dried under
nitrogen gas and kept under high vacuum for 1 h. The
lipid was suspended in 10 mM phosphate buffer (pH 7.4)
and freeze-thawed 5 times in a MeOH bath. The multilamellar vesicle suspension was transferred into a Lipofast
Apparatus (Avestin, Ottawa, Canada) and passed thorougha polycarbonate membrane (pore size 100 nm) 19 times toobtain unilamellar liposomes (10 mg mL–1). The antioxidant
activity of the sample to be tested in the liposome system wasmeasured by the method of Esaki et al. (1997). The test
samples were dissolved in 100 μL ethanol (EtOH). In thecontrol test, 100 μL EtOH was used instead of the sample
solution. The amount of thiobarbituric acid-reacting substancewas determined by measuring the absorbance at 535 nm.

Preparation of phlorotannin–soybean protein complex

1.5

10
20
Retention time (min)30

P-5 P-4

P-6
P-1

P-3

0
200

300
Wavelength (nm)
400
0

10
20
Retention time (min)30

Fig. 2 Reversed-phase three-dimensional high-performance liquid
chromatography (RP-3D-HPLC) of crude phlorotannins from theLaminariaceous brown algae Eisenia bicyclis (a), Ecklonia cava (b)and Ecklonia kurome (c). P-1 Phloroglucinol, P-2 phloroglucinol
tetramer, P-3 eckol, P-4 phlorofucofuroeckol A, P-5 dieckol, P-6 8,8′bieckol

708

autoclaved at 121°C for 20 min. After filtration, thesoybean protein extract (500 mL) was mixed with
100 mL of the crude phlorotannins from Eisenia bicyclis
in water (10 mg/mL), and the pH of the mixture wasadjusted to 4.5 with 2 N HCl. The phlorotannin–soybean
protein complex that precipitated in the acidic solution waslyophilized. The amount of phlorotannins conjugated tosoybean protein was determined by RP-3D-HPLC. The
conjugated amount (%) was calculated using the following
formula:Conjugation ratio (%) = (1− the amount of phlorotannins
detected in the supernatant of the phlorotannin–soybean
protein complex / the initial content of each compounds inthe crude phlorotannins) × 100.
The DPPH-radical scavenging activity of the complex
was measured using the method of Huang et al. (2004). In
the control test, lyophilized soybean protein extract wasused instead of the sample.

J Appl Phycol (2008) 20:705–711

identified by comparing their retention times with those of
purified phlorotannins and their on-line ultraviolet spectra.
Within 30 min, a distinct separation of each of thecomponents in the brown algal extracts was obtained by alinear gradient system of 30–100% MeOH (Fig. 2). Eachindividual phlorotannin was purified by silicic acid column
chromatography. The purity of each oligomer in thisexperiment exceeded 90%.

Results
The yield of crude phlorotannins from Eisenia bicyclis,
Ecklonia cava and Ecklonia kurome was about 3% of thealgal powder (dry weight) for all three algae. The crude
phlorotannins from the brown algae were analyzed by TLC
and RP-3D-HPLC. As shown in Fig. 1, five to six spots
detected in all three algal fractions were stained by paprika
pigment, indicating the presence of antioxidant substances.
Judging from previous reports (Nakamura et al. 1996;
Shibata et al. 2004, 2006b), the spots can be identified asfollows: phloroglucionl (P-1), unknown phloroglucinol
tetramer (P-2), eckol (P-3), phlorofucofuroeckol A (P-4),
dieckol (P-5) and 8,8′-bieckol (P-6). In RP-3D-HPLC
analysis, peaks in the fractions of crude phlorotannins were

Fig. 3 Antioxidant activities of phlorotannins, terrestrial polyphenols
and antioxidant vitamins in the liposome system. Lipid peroxidation
was induced by 4 mM 2, 2′-azobis (2-amidinopropane) dihydrochloride. The analytical data are presented as mean ± SD (n=5)

Fig. 4a,b Radical scavenging activities of phlorotannins, terrestrial
polyphenols and antioxidant vitamins. a 1-Diphenyl-2-picrylhydrazyl(DPPH)-radical, b superoxide anion radical. All analytical data are themeans of three determinations

J Appl Phycol (2008) 20:705–711

709

Table 1 Conjugation of phlorotannins to soybean protein. Reversedphase three-dimensional high-performance liquid chromatography(RP-3D-HPLC) conditions are described in Materials and methods.
All analytical data in the table are the mean of three determinations
CompoundMolecular
weightConjugation(%)

Eckol (P-3)
Unknown phloroglucinol tetramer(P-2)
Phlorofucofuroeckol A (P-4)
Dieckol (P-5)
8,8′-Bieckol (P-6)

372
478

27.5
98.2

602
742
742

100
100
100

Antioxidant activity of phlorotannins in the liposome
systemFigure 3 shows the inhibitory effects of phlorotannins,terrestrial polyphenols and antioxidant vitamins on phospholipid peroxidation in a liposome system initiated by
2,2′-azobis (2-amidinopropane) dihydrochloride. At a final
concentration of 1 μM, eckol, phlorofucofuroeckol A,
dieckol and 8,8′-bieckol each showed about 30% inhibitionof phospholipid peroxidation. Thus, the oligomeric phloroglucinols were found to possess potent antioxidant
activity, comparable in magnitude to that of well-known
antioxidants such as catechins and vitamins.
Radical scavenging activities of phlorotannins

Fig. 5 DPPH-radical scavenging activity of phlorotannin–soybean protein complex. The
complex was steeped in acetate
buffer (pH 4.5) overnight atroom temperature before being
homogenized and centrifuged.
The supernatant was mixed with
the DPPH in EtOH. All analytical data are the means of three
determinations

DPPH-radical scavenging activity (%)

A further experiment was carried out using the DPPHradical and the superoxide anion radical scavenging assays

to determine the antioxidant activity of the phlorotannins.
With the exception of phloroglucinol and resveratrol, thephlorotannins, terrestrial polyphenols and antioxidant vitamins scavenged the DPPH-radical in a dose-dependent
manner (Fig. 4a). The EC50 values of the phlorotannins
eckol, phlorofucofuroeckol A, dieckol and 8,8′-bieckol
were 26, 12, 13 and 15 μM, respectively. In contrast, thoseof catechin, EGCG, ascorbic acid and α-tocopherol were32, 7.4, 30, and 52 μM, respectively. The phlorotannins
were potent free radical scavengers and about twice aseffective as catechin, ascorbic acid and α-tocopherol.
Figure 4b shows the superoxide anion scavenging activitiesof the phlorotannins, terrestrial polyphenols and antioxidant
vitamins. The EC50 values of eckol, phlorofucofuroeckol A,
dieckol, 8,8′-bieckol, catechin, EGCG, resveratrol, ascorbic
acid and α-tocopherol were 107, 8.4, 7.6, 6.5, 5.2, 2.1, 21,62, and 12 μM, respectively. With the exception of eckol,
the phlorotannins were found to have very potent superoxideanion radical scavenging activity, being about 2–10 times
more active than ascorbic acid and α-tocopherol.
Phlorotannin–soybean protein complex
In order to use phlorotannins as functional food ingredient, acomplex of crude phlorotannins and soybean protein was
prepared. As shown in Table 1, phlorotannins of highmolecular weight—the pentamer and hexamers of phloroglucinol—showed pronounced affinity for the soybean
protein. About 5 g phlorotannin–soybean protein complex
was obtained from 1 g crude phlorotannins and 200 g
defatted soybean. The DPPH-radical scavenging activity of
the complex was about four times stronger than that of thelyophilized soybean protein extract alone (Fig. 5).

100

80
60

Phlorotannins-soyben protein complex
40
20
0
1

10

100

Concentration (mg/mL)
Lyophilized soybean protein extract

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J Appl Phycol (2008) 20:705–711

Discussion

References

A main target of free radical damage in the body is the cell
membrane, which contains abundant unsaturated lipids. The
liposome has been used extensively as a model of the cell
membrane for in vitro biochemical research. Therefore, theunilamellar liposome lipid/aqueous system was used toevaluate the antioxidative properties of phlorotannins. In
the present study, the oligomeric phloroglucinols were
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or supplement with anti-inflammatory activity.

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