JOURNAL OF FUNCTIONAL FOODS 5 ( 20 1 3 ) 20 1 9–20 2 3

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The anti-inflammatory effects of phlorotannins

from Eisenia arborea on mouse ear edema
by inflammatory inducers

Yoshimasa Sugiuraa,*, Ryusuke Tanakaa, Hirotaka Katsuzakib, Kunio Imaib,

Teruo Matsushitaa
a
The Laboratory of Food Function and Food Biochemistry, Department of Food Science and Technology, National Fisheries University,
2-7-1 Nagata-Honmachi, Shimonoseki, Yamaguchi 759-6595, Japan
b
Laboratory of Bioorganic Chemistry, Graduate School of Bioresources, Mie University, Kurimamachiya-cho 1577, Tsu, Mie 514-8507, Japan

A R T I C L E I N F O A B S T R A C T

Article history: The anti-allergic and anti-inflammatory effects of phlorotannins (seaweed polyphenols)
Received 30 April 2013 from the brown alga Eisenia arborea using in vitro experiments has already been reported.
Received in revised form Therefore, in this study, these effects were examined in vivo using mice. When ear edema
12 August 2013 was induced in ICR mice by three sensitizers (arachidonic acid (AA), 12-O-tetradecanoyl-
Accepted 21 August 2013 phorbol-13-acetate (TPA) and oxazolone (OXA)), four phlorotannins tested (eckol, 8,8’-biec-
Available online 13 September 2013 kol, phlorofucofuroeckol (PFF)-A and PFF-B) inhibited the ear edema after spreading of 0.01
or 0.1 mg onto the mouse ear. The effects of the phlorotannins on the ear edema after AA
Keywords: and OXA treatments tended to be stronger than after TPA treatment, and the inhibitory
Anti-allergy effects against each sensitizer were similar to epigallocatechin gallate, a typical inhibitor.
Anti-inflammation This is the first report that phlorotannins exhibit anti-allergic and anti-inflammatory
Eisenia arborea
effects in in vivo experiments, and inhibit OXA-induced delayed-type (type IV) allergic
Mouse ear edema
reactions.
Phlorotannin
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction phorbol-13-acetate (TPA) and oxazolone (OXA)) (Huang, Ghai,

There are many reports regarding the anti-allergic and anti-
inflammatory effects of terrestrial plant polyphenols such
as curcumin, epigallocatechin gallate, luteolin and quercetin
in not only in vitro but also in vivo experiments. These terres-
trial plant polyphenols also inhibit mouse ear edema induced
by sensitizers (arachidonic acid (AA), 12-O-tetradecanoyl-
& Ho, 2004; Kim, Son, Chang, & Kang, 2004; Ueda, Yamazaki, &
Yamazaki, 2002).
Seaweed polyphenols (phlorotannins) such as eckol, diec-
kol, 6,6’- or 8,8’-bieckol and phlorofucofuroeckol (PFF)-A are
well known (Ragan & Glombitza, 1986). Phlorotannins appear
to possess anti-allergic and anti-inflammatory effects similar
to terrestrial plant polyphenols. For example, brown algal

* Corresponding author. Tel.: +81 83 286 5111; fax: +81 83 286 7434.
E-mail address: ysugiura@fish-u.ac.jp (Y. Sugiura).
Abbreviations: AA, arachidonic acid; EGCg, epigallocatechin gallate; OXA, Oxazolone; PFF, Phlorofucofuroeckol; TPA, 12-O-
tetradecanoylphorbol-13-acetate
1756-4646/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jff.2013.08.010
2020 JOURNAL OF FUNCTIONAL FOODS
phlorotannins inhibit activities of phospholipase A2, lipoxy-
genase, cyclooxygenase-2 and hyaluronidase which are in-
volved in allergic reactions (Shibata, Nagayama, Tanaka,
Yamaguchi, & Nakamura, 2003), and suppress cyclooxygen-
ase-2 protein gene expression via the mitogen-activated pro-
tein kinase (MAPK) pathway (Ryu, Li, Qian, Kim, & Kim, 2009).
We have previously reported the anti-allergic effects of Eisenia
arborea phlorotannins using in vitro experiments (Sugiura
et al., 2007; Sugiura et al., 2008b; Sugiura et al., 2009). In
in vivo experiments, the anti-allergic effect of dried E. arborea
powder was observed using Brown Norway (BN) rats, an aller-
gic model (Sugiura, Matsuda, Okamoto, Kakinuma, & Amano,
2008a). However, the reports for those phlorotannins were
from in vitro experiments, and the study of BN rats did not ap-
pear to show that the active compounds were phlorotannins
or polysaccharides like alginate and fucoidan.
Therefore, in this study, the anti-inflammatory effects of
phlorotannins, as purified active compounds, were examined
in vivo using ICR mice. As sensitizers of mouse ear edema, AA
involved in lipoxygenase (LOX) activity, TPA inducing cycloox-
ygenase (COX)-2 expression and OXA inducing delayed-type
(type IV) allergic reactions were used (Kujubu, Fletcher, Var-
num, Lim, & Herschman, 1991; Nakano, 1977; Young et al.,
1984), and the effects of the phlorotannins against the three
different types of inflammation were investigated.
2. Materials and methods
2.1. Materials
E. arborea was collected from the coast of Mugizaki in Mie pre-
fecture, Japan. The powdering of the collected alga was car-
ried out based on the procedure of Sugiura et al. (2008a).
Phlorotannins (eckol, 8,8’-bieckol, phlorofucofuroeckol (PFF)-
A and PFF-B) (Fig. 1) tested in this study were purified in
2009 by methanol/chloroform (M/C) extraction and HPLC frac-
tionation from the algal powder (Sugiura et al., 2009), and
have been stored at 80 °C in methanol since that time. Since
degradation of the phlorotannins was suspected due to the
long storage period, HPLC analysis and a DPPH radical scav-
enging assay (Sugiura et al., 2008b) were conducted. No degra-
dation was observed (data not shown).
Epigallocatechin gallate (EGCG, Sigma–Aldrich, St. Louis,
MO, USA) was used as a natural typical inhibitor, since EGCG
is well known to possess anti-allergic and anti-inflammatory
effects (Liang et al., 2002; Yoshino, Kawaguchi, Yamazaki, &
Sano, 2010).
2.2. Assays by mouse ear edema
2.2.1. Animals
As an ear edema model, ICR mice were used. The mice (male,
4 weeks old) were purchased from Kyudo Co., Ltd. (Tosu, Saga,
Japan) and housed in individual cages kept at 23–26 °C under
a 12 h light/dark cycle until being tested. Food (MF, KBT Orien-
tal Co. Ltd., Tosu, Saga, Japan) and tap water were freely avail-
able. All of the animal experiments were performed with
permission of the ‘‘Committee for Use and Care of Laboratory
Animals’’ of the National Fisheries University, and in compli-
5 ( 2 0 1 3 ) 2 0 1 9 –2 0 2 3
ance with the ‘‘Guideline for Animal Experiments in Research
Institutes under the jurisdiction of the Ministry of Agriculture,
Forestry and Fisheries’’.
2.2.2. Arachidonate (AA)
AA-induced ear edema was induced by the method of Young
et al. (1984) with some modifications. Ten microlitres of AA
(Wako; 12.5 mg/mL in acetone, stored at 20 °C until used)
and 5 lL of sample (0.01 mg or 0.1 mg/5 lL) dissolved in meth-
anol were mixed and spread on a mouse ear. One hour later,
ear swelling was determined with a thickness gauge (547 ser-
ies, Mitutoyo Corporation, Kawasaki, Kanagawa, Japan). The
inhibition ratio of the ear swelling was calculated according
to the following formula:
Inhibition ratioð%Þ ¼ ½1  ðT  T0 Þ=ðC  C0 Þ  100
where C0 is ear thickness without test sample before spread-
ing AA; C is the ear thickness without test sample after
spreading AA; T0 is the ear thickness with test sample before
spreading AA and T is the ear thickness with test sample after
spreading AA.
2.2.3. 12-O-tetradecanoylphorbol-13-acetate (TPA)
TPA-induced ear edema was induced by the method of Young
et al. (1984) with some modifications. Immediately before use,
a stock solution of TPA (Wako; 800 lg/mL in acetone, stored at
20 °C until used) was diluted to 80 lg/mL with acetone. Ten
microliters of TPA and 5 lL of sample (0.01 mg or 0.1 mg/5 lL)
were mixed and spread on a mouse ear. Four hours later, ear
swelling was determined with a thickness gauge. The inhibi-
tion ratio of the ear swelling was calculated as described
above.
2.2.4. Oxazolone (OXA)
OXA-induced ear edema was induced by the method of Yosh-
ino et al. (2010) with some modifications. The OXA solution
(Sigma–Aldrich) was stored at 20 °C until used. Fifty microli-
ters of 1% OXA dissolved in ethanol was spread on the
abdominal region of a mouse whose abdominal hair was care-
fully shaved with an animal shaver (Natsume Seisakusho Co,
Ltd., Tokyo, Japan) under anesthesia with diethyl ether. Five
days later, 10 lL of 0.5% OXA dissolved in acetone and 5 lL
of sample (0.01 mg or 0.1 mg/5 lL) were mixed and spread
on a mouse ear. Twenty-four hours later, ear swelling was
determined with a thickness gauge. The inhibition ratio of
the ear swelling was calculated as described above.
2.3. Statistical analysis
The inhibition ratios were calculated from the results of qua-
druplicate (n = 4) experiments. Data are expressed as
means ± SD. The statistical analysis of the multiple compari-
son was done by Dunnett’s test. P < 0.05 was considered sta-
tistically significant.
3. Results and discussion
As shown in Table 1, the four E. arborea phlorotannins (eckol,
8,8’-bieckol, PFF-A and PFF-B) and the methanol/chloroform
(M/C) extract that is a condensed crude solution containing
 JOURNAL OF FUNCTIONAL FOODS 5 ( 2 01 3 ) 20 1 9–20 2 3 2021

OH
OH OH
O OH
OH O OH
OH O OH HO O O OH
O OH HO O OH
HO O
OH
HO O OH
OH

Eckol 8,8'-bieckol

OH OH

OH O OH OH
OH O OH
O OH HO O OH
OH
O O O O O
HO OH
OH OH
O OH
HO HO

Phlorofucofuroeckol-A Phlorofucofuroeckol-B

Fig. 1 – The chemical structures of phlorotannins from Eisenia arborea.

Table 1 – Inhibition of AA-induced ear edema by topically administered phlorotannins.

Samples eckol 8,8’-bieckol PFF-A PFF-B M/C EGCG

Inhibition ratio (%) 0.01 (mg/mouse) 34.4 ± 23.8 39.5 ± 12.2 43.5 ± 14.5 47.6 ± 10.7 25.9 ± 14.3 35.5 ± 27.5
0.1 (mg/mouse) 49.2 ± 17.5 79.8 ± 17.5 67.7 ± 15.4 78.2 ± 8.5 57.1 ± 6.9 59.1 ± 14.4
Values are means ± standard deviations (n = 4). The inhibitory effects of phlorotannins were comparable to that of EGCG (P > 0.05).
Abbreviations: AA, arachidonic acid; PFF, phlorofucofuroeckol; M/C, methanol/chloroform extract; EGCg, epigallocatechin gallate.

these four phlorotannins inhibited AA-induced ear edema.
The inhibitory effects of phlorotannins were comparable
(P > 0.05) to that of EGCG at both 0.01 and 0.1 mg/mouse. At
0.01 mg/mouse, all samples tested inhibited ear edema to
some degree, and 8,8’-bieckol at 0.1 mg/mouse exerted the
strongest inhibition up to 79.8%. The inhibitory effects of
8,8’-bieckol (79.8%), PFF-A (67.7%) and PFF-B (78.2%) at
0.1 mg/mouse (P > 0.05) tended to be stronger than EGCG
(59.1%). The inhibition ratio of EGCG at 0.1 mg/mouse was
comparable to the results of Yoshino and Miyauchi (2007),
who showed that EGCG at 0.13 mg/mouse suppressed AA-in-
duced mouse ear edema up to about 46%.
As shown in Table 2, the four phlorotannins and the M/C
extract inhibited TPA-induced ear edema. The inhibitory ef-
fects of phlorotannins were comparable to that of EGCG at
both 0.01 and 0.1 mg/mouse (P > 0.05). At 0.01 mg/mouse, all
samples tested inhibited ear edema to some degree, and
PFF-B at 0.1 mg/mouse exerted the strongest inhibition up
to 56.1%. The inhibitory effects of the sample tested on TPA-
induced ear edema tended to be weaker than AA-induced
ear edema. At 0.1 mg/mouse, PFF-B and EGCG (54.4%) exhib-
ited over 50% inhibition. In this study, the inhibition ratio of
EGCG at 0.1 mg (0.22 lmol)/mouse was 54.4%. Because there
is a report that EGCG at 1 lmol suppressed TPA-induced
mouse ear edema up to 85.2% (Liang et al., 2002), our result with
EGCG is consistent with this report. Thus, since the phlorotan-
nins from E. arborea inhibited AA- and/or TPA-induced mouse
ear edema to a similar degree as EGCG, whose inhibitory effect
is well-known, the phlorotannins tested might be active toward
the immediate inflammatory reactions.
The inhibitory effects of phloroannins tested on TPA-in-
duced mouse ear edema tended to be weaker than that of
AA-induced mouse ear edema (Table 1 and 2). AA-induced
ear edema is involved in LOX related activity (Young et al.,
1984). On the other hand, in TPA-induced inflammation,
COX-2 gene is rapidly expressed (Kujubu et al., 1991). The
phlorotannins tested exhibited inhibitory effects on COX-2
and LOX activities. However, since the concentrations that
exhibited the inhibitory effects (IC50 values) of the phlorotan-
nins are lower for LOX inhibition than for COX-2 inhibition,
the inhibitory effects of phlorotannins on COX-2 activity are
weaker than those on LOX activity (Sugiura et al., 2009). Thus,
the reason why the inhibitory effects of phloroannins tested
on TPA-induced inflammation involved in COX-2 gene
expression tended to be weaker than that of AA-induced
inflammation involved in LOX activity could be attributed to
the different inhibitory strength of the phlorotannins on
the two enzyme activities. Because phlorotannin-inhibited
2022 JOURNAL OF FUNCTIONAL FOODS 5 ( 2 0 1 3 ) 2 0 1 9 –2 0 2 3

Table 2 – Inhibition of TPA-induced ear edema by topically administered phlorotannins.

Samples eckol 8,8’-bieckol PFF-A PFF-B M/C EGCG

Inhibition ratio (%) 0.01 (mg/mouse) 20.4 ± 15.0 21.3 ± 11.5 14.8 ± 13.4 23.1 ± 14.0 26.6 ± 7.8 25.7 ± 23.9
0.1 (mg/mouse) 38.0 ± 5.6 49.1 ± 18.5 44.4 ± 20.6 56.1 ± 11.2 38.7 ± 4.6 54.4 ± 20.0
Values are means ± standard deviations (n = 4). The inhibitory effects of phlorotannins were comparable to that of EGCG (P > 0.05).
Abbreviations: TPA, 12-O-tetradecanoylphorbol-13-acetate; PFF, phlorofucofuroeckol; M/C, methanol/chloroform extract; EGCg, epigallocatechin
gallate.

Table 3 – Inhibition of OXA-induced ear edema by topically administered phlorotannins.

Samples eckol 8,8’-bieckol PFF-A PFF-B M/C EGCG

Inhibition ratio (%) 0.01 (mg/mouse) 44.9 ± 25.3 38.9 ± 15.0 37.7 ± 21.2 35.9 ± 22.6 32.5 ± 11.3 47.2 ± 25.3
0.1 (mg/mouse) 65.3 ± 20.7 71.3 ± 6.8 76.6 ± 9.5 71.6 ± 9.4 62.5 ± 11.3 75.7 ± 7.2
Values are means ± standard deviations (n = 4). The inhibitory effects of phlorotannins were comparable to that of EGCG (P > 0.05).
Abbreviations: OXA, oxazolone; PFF, phlorofucofuroeckol; M/C, methanol/chloroform extract; EGCg, epigallocatechin gallate.

COX-2 gene expression contributed to inhibition of the MAPK
pathway (Ryu et al., 2009), the inhibitory effects of phlorotan-
nins on TPA-induced inflammation could be attributed to
inhibition of COX-2 gene expression rather than inhibition
of COX-2 activity.
As shown in Table 3, the four phlorotannins and the M/C
extract inhibited OXA-induced ear edema. The inhibitory ef-
fects of the phlorotannins were comparable to that of EGCG
at both 0.01 and 0.1 mg/mouse (P > 0.05). At 0.01 mg/mouse,
all samples tested inhibited ear edema to some degree, and
PFF-A at 0.1 mg/mouse exerted the strongest inhibition up
to 76.6%. The inhibitory effects of the samples tested on
OXA-induced ear edema were comparable to AA-induced
ear edema, and tended to be stronger than TPA-induced ear
edema. At 0.1 mg/mouse, all samples exhibited over 60% inhi-
bition. These data at 0.1 mg/mouse are comparable to the
finding that EGCG at 0.13 mg/mouse suppressed OXA-induced
mouse ear edema up to 88.0% (Suzuki, Yoshino, Maeda-
Yamamoto, Miyase, & Sano, 2000). Thus, phlorotannins are
as effective as EGCG against OXA-induced inflammation that
is involved in delayed type (type IV) allergic reactions (Nak-
ano, 1977). The present data for the inhibitory effects of
phlorotannins on OXA-induced type IV allergic reactions are
novel and very useful information for the health application
of marine food products.
Thus, phlorotannins inhibited inflammatory reactions in
mouse ear edema. But, since orally-administered EGCG and
luteolin suppressed AA, TPA and OXA-induced mouse ear
edema (Ueda et al., 2002; Yoshino et al., 2010), the anti-inflam-
matory effects of phlorotannins on mouse ear edema needs
to be examined in mice orally administered, considering
application to food utilization. Also, further studies for
in vivo kinetics and intestinal absorption on phlorotannins
are needed.
4. Conclusions
In summary, phlorotannins (eckol, 8,8’-bieckol, PFF-A and
PFF-B) from E. arborea exhibited anti-inflammatory effects
induced by AA, TPA, and OXA in mouse ear edema. These
results represent novel knowledge using experimental ani-
mals to determine anti-allergic and anti-inflammatory effects
of marine food products.
Acknowledgements
This work was supported by JSPS KAKENHI Grant Number
23880036. We thank Dr. D. A. Coury (Phycal Inc., Highland
Heights, OH, USA) for reviewing the English and for helpful
suggestions regarding the manuscript.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.jff.2013.08.010.
R E F E R E N C E S
Huang, M.-T., Ghai, G., & Ho, C.-T. (2004). Inflammatory process
and molecular targets for antiinflammatory nutraceuticals.
Comprehensive Reviews in Food Science and Food Safety, 3, 127–139.
Kim, H.-P., Son, K.-H., Chang, H.-W., & Kang, S.-S. (2004). Anti-
inflammatory plant flavonoids and cellular action
mechanisms. Journal of Pharmacological Sciences, 96, 229–245.
Kujubu, D. A., Fletcher, B. S., Varnum, B. C., Lim, R. W., &
Herschman, H. R. (1991). TIS10, a phorbol ester tumor
promoter-induciblem RNA from Swiss 3T3 cells, encodes a
novel prostaglandin synthase/cyclooxygenase homologue. The
Journal of Biological Chemistry, 266, 12866–12872.
Liang, Y.-C., Tsai, D.-C., Lin-Shiau, S.-Y., Chen, C.-F., Ho, C.-T., &
Lin, J.-K. (2002). Inhibition of 12-O-tetradecanoylphorbol-13-
acetate-induced inflammatory skin edema and ornithine
decarboxylase activity by theaflavin-3,3-digallate in mouse.
Nutrition and Cancer, 42, 217–223.
Nakano, Y. (1977). Antigenic competition in the induction of
contact sensitivity in mice. Immunology, 33, 167–178.
Ragan, M. A., & Glombitza, K. W. (1986). Phlorotannins, brown
algal polyphenols. Progress in Phycological Research, 4, 129–241.
Ryu, B., Li, Y., Qian, Z.-J., Kim, M.-M., & Kim, S.-K. (2009).
Differentiation of human osteosarcoma cells by isolated
phlorotannins is subtly linked to COX-2, iNOS, MMPs, and
 JOURNAL OF FUNCTIONAL FOODS
MAPK signaling: Implication for chronic articular disease.
Chemico-Biological Interactions, 179, 192–201.
Shibata, T., Nagayama, K., Tanaka, R., Yamaguchi, K., &
Nakamura, T. (2003). Inhibitory effects of brown algal
phlorotannins on phospholipase A2s, lipoxygenases and
cyclooxygenases. Journal of Applied Phycology, 15, 61–66.
Sugiura, Y., Matsuda, K., Okamoto, T., Kakinuma, M., & Amano, H.
(2008a). Anti-allergic effects of the brown alga Eisenia arborea
on Brown Norway rats. Fisheries Science, 74, 180–186.
Sugiura, Y., Matsuda, K., Okamoto, T., Yamada, Y., Imai, K., Ito, T.,
Kakinuma, M., & Amano, H. (2009). The inhibitory effects of
components from a brown alga, Eisenia arborea, on
degranulation of mast cells and eicosanoid synthesis. Journal
of Functional Foods, 1, 387–393.
Sugiura, Y., Matsuda, K., Yamada, Y., Imai, K., Kakinuma, M., &
Amano, H. (2008b). Radical scavenging and hyarulonidase
inhibitory avtivitise of phlorotannins from the edible brown
alga Eisenia arborea. Food Science and Technology Research, 14,
595–598.
Sugiura, Y., Matsuda, K., Yamada, Y., Nishikawa, M., Shioya, K.,
Katsuzaki, H., Imai, K., & Amano, H. (2007). Anti-allergic
5 ( 2 01 3 ) 20 1 9–20 2 3 2023
phlorotannins from the edible brown alga, Eisenia arborea. Food
Science and Technology Research, 13, 54–60.
Suzuki, M., Yoshino, K., Maeda-Yamamoto, M., Miyase, T., & Sano,
M. (2000). Inhibitory effects of tea catechins and O-methylated
derivatives of (-)-epigallocatechin-3-O-gallate on mouse type
IV allergy. Journal of Agricultural and Food Chemistry, 48,
5649–5653.
Ueda, H., Yamazaki, C., & Yamazaki, M. (2002). Luteolin as an anti-
inflammatory and anti-allergic constituent of Perilla frutescens.
Biological & Pharmaceutical Bulletin, 25, 1197–1202.
Yoshino, K., Kawaguchi, T., Yamazaki, K., & Sano, M. (2010).
Preventive effects of (-)-epigallocatechin-3-O-gallate on mouse
type IV allergy induced by oxazolone and its antiinflammatory
activities. Journal of Technology and Education, 17, 57–65.
Yoshino, K., & Miyauchi, Y. (2007). Preventive effects of various
antioxidants on mouse contact dermatitis. Journal of Technology
and Education, 14, 37–44 (in Japanese).
Young, J. M., Spires, D. A., Bedord, C. J., Wagner, B., Ballaron, S. J., &
De Young, L. M. (1984). The mouse ear inflammatory response
to topical arachidonic acid. Journal of Investigative Dermatology,
82, 367–371.