Toxicology in Vitro 23 (2009) 1123–1130

Contents lists available at ScienceDirect

Toxicology in Vitro
journal homepage: www.elsevier.com/locate/toxinvit

Effect of phlorotannins isolated from Ecklonia cava on melanogenesis and their

protective effect against photo-oxidative stress induced by UV-B radiation
Soo-Jin Heo a, Seok-Chun Ko b, Seon-Heui Cha b, Do-Hyung Kang a, Heung-Sik Park a, Young-Ung Choi a,
Daekyung Kim c, Won-Kyo Jung d, You-Jin Jeon b,*
a
Marine Living Resources Research Department, Korea Ocean Research and Development Institute, Ansan 426-744, Republic of Korea
b
Faculty of Applied Marine Science, Jeju National University, Jeju 690-756, Republic of Korea
c
Jeju Center, Korea Basic Science Institute, Jeju 690-756, Republic of Korea
d
Department of Marine Life Science and Marine Life Research Center, Chosun University, Gwangju 501-759, Republic of Korea

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, three kinds of phlorotannins, marine algal polyphenol, were isolated from a brown
Received 3 February 2009 alga Ecklonia cava, and their inhibitory effect on melanogenesis as well as the protective effect against
Accepted 23 May 2009 photo-oxidative stress induced by UV-B radiation was investigated. The effect on melanogenesis was
Available online 31 May 2009
evaluated via the inhibitory effects of tyrosinase and melanin synthesis. Among the phlorotannins, diec-
kol showed higher effect than that of the other phlorotannins in the both assays; especially the value of
Keywords: dieckol in the tyrosinase inhibition assay was relatively higher than that of a commercial tyrosinase
Phlorotannin
inhibitor (kojic acid). The UV-B protection effect was evaluated via DCFH-DA, MTT, comet assays, and
Dieckol
Ecklonia cava
morphological changes in fibroblast. Intracellular ROS induced by UV-B radiation was reduced by the
Melanogenesis addition of phlorotannins and cell viability was dose-dependently increased. Moreover, dieckol demon-
UV-B protection strated strong protective properties against UV-B radiation-induced DNA damage via damaged tail inten-
sity and morphological changes in fibroblast. Hence, these results indicated that dieckol isolated from E.
cava has potential whitening effects and prominent protective effects on UV-B radiation-induced cell
damages, which might be used in pharmaceutical and cosmeceutical industries.
Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.

1. Introduction lished that overexposure to UV radiation provokes acute sunburn

Cosmetics are commercially available products that are used to
improve the appearance of the skin. In recent years, the number of
women concerning about whiter skin complexion, especially in
Asia has increased dramatically (Tengamnuay et al., 2006). Mela-
nin is the major pigment responsible for the color of human skin.
It may be overproduced with chronic sun exposure, melasma, or
other hyperpigmentation diseases. Therefore, a number of depig-
menting agents have been developed in cases of undesirable skin
discoloration. Tyrosinase, a copper-containing monooxygenase, is
a key enzyme that catalyzes melanin synthesis in melanocytes. It
is catalyzes the hydroxylation of tyrosine into dihydroxyphenylal-
anine (DOPA) and other intermediates (Sturm et al., 2001; Kajiwara
et al., 2006; Wang et al., 2006). Thus, inhibition of tyrosinase activ-
ity or its production can prevent melanogenesis.
Skin is the preferred target of oxidative stress as continuously
exposed to ultraviolet (UV) radiation from sunlight and environ-
mental oxidizing pollutants (Thiele et al., 1997). It is well estab-
* Corresponding author. Tel.: +82 64 754 3475; fax: +82 64 756 3493.
E-mail address: youjinj@cheju.ac.kr (Y.-J. Jeon).
reaction, which clinically manifests itself as erythema. Chronically
irradiated skins by UV radiation are associated with abnormal
cutaneous reactions such as epidermal hyperplasia, accelerated
breakdown of collagen, and inflammatory responses (Longstreth
et al., 1998; Marrot et al., 2001; Tanaka et al., 2007). UV radiation
has a strong oxidative component, and photo-oxidative stress has
been directly linked to the onset of skin photodamage (Fuchs,
1998; Caddeo et al., 2008). Hence, regular intake of dietary antiox-
idants or treatment of the skin with products containing antioxi-
dant ingredients may be a useful strategy for preventing
UV-induced damages.
Marine algae produce a great variety of secondary metabolites
possessing many different skeletal types and biological activities
(Heo et al., 2005; Heo and Jeon, 2008; Sabry et al., 2005). Although
these marine algae expose to the adverse environmental condi-
tions such as light and high oxygen concentrations that lead to
the formation of free radicals, and other strong oxidizing agents,
they do not affect any serious photodynamic damage in vivo. This
fact suggests that marine algae like photosynthesizing plants have
antioxidative mechanisms and compounds which act as antioxi-
dant agents (Dykens et al., 1992; Jimenez-Escrig et al., 2001).
Further, it is well established that brown algae contain phenolic

0887-2333/$ - see front matter Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.tiv.2009.05.013
1124 S.-J. Heo et al. / Toxicology in Vitro 23 (2009) 1123–1130
compounds with strong antioxidant activity. Marine algal polyphe-
nols, known as phlorotannins, which have only been found to exist
within brown algae, are restricted to polymers of phloroglucinol
(1,3,5-trihydroxybenzene). It has been reported that phlorotannins
have several biological functions including anti-plasmin inhibitors,
anti-allergic, antibacterial and antioxidant activities (Nakayama
et al., 1989; Nakamura et al., 1996; Nagayama et al., 2002; Sugiura
et al., 2006).
Ecklonia cava, a kind of brown alga, is plentifully produced in
Jeju Island in Korea, and is utilized as food ingredient, animal feed,
fertilizer and medicine. In addition, E. cava has a variety of com-
pounds including carotenoid, fucoidans, and phlorotannins show-
ing different biological activities. In a previous study (Ahn et al.,
2007), we isolated three kinds of phlorotannins from E. cava,
including phloroglucinol, eckol and dieckol, which showed poten-
tial antioxidant activity. Accordingly, the present study aimed to
isolate a marine natural phlorotannins from E. cava and evaluated
the effects of the phlorotannins on melanogenesis and their photo-
protective effect on the cell damage induced by UV-B radiation.
2. Materials and methods
2.1. Materials
The marine alga E. cava was collected along the coast of Jeju Is-
land, Korea, between October 2007 and March 2008. The samples
were washed three times with tap water to remove the salt, epi-
phytes, and sand attached to the surface. Then carefully rinsed
with fresh water, and maintained in a medical refrigerator at
20 °C. Thereafter, the frozen samples were lyophilized and
homogenized with a grinder prior to extraction.
2.2. Extraction and isolation
The phlorotannins were isolated as previously described by Ahn
et al. (2007) with slight modifications. Briefly, the dried E. cava
powder (500 g) was extracted three times with 80% MeOH and
then was filtered. The filtrate was evaporated at 40 °C to obtain
the methanol extract. After, the extract was suspended on distilled
water, and partitioned with ethyl acetate. The ethyl acetate frac-
tion was mixed with celite. The mixed celite was dried and packed
into a glass column, and eluted in the order of hexane, methylene
chloride, diethyl ether, and methanol. The diethyl ether fraction
was further purified by sephadex LH-20 column chromatography
using stepwise gradient chloroform/methanol (2/1 ? 0/1) solvents
system. The phloroglucinol, eckol, and dieckol were purified by
high performance liquid chromatography (HPLC) using a Waters
HPLC system equipped with a Waters 996 photodiode array detec-
tor and C18 column (J’sphere ODS-H80, 150  20 mm, 4 lm; YMC
Co.) by stepwise elution with methanol–water gradient (UV range:
230 nm, flow rate: 0.8 ml/min). Finally, the purified compounds
were identified by comparing their 1H and 13C NMR data to the lit-
erature report. The chemical structures of the three phlorotannins
are indicated in Fig. 1.
2.3. Inhibitory effect of mushroom tyrosinase
Tyrosinase inhibitory activity was performed according to the
method of Vanni et al. (1990) with slight modifications. The reac-
tion mixture contains 140 ll of 0.1 M phosphate buffer (pH 6.5),
40 ll of 1.5 mM L-tyrosine and 10 ll of samples. Then, 10 ll of
mushroom tyrosinase (2100 units/ml) solution was added and
the reaction was incubated at 37 °C for 12 min. After incubation,
the amount of dopachrome produced in the reaction mixture was
determined as the optical density at 490 nm in a microplate reader.
2.4. Inhibitory effect of melanin synthesis
Melanin contents were measured according to the method de-
scribed by Tsuboi et al. (1998) with slight modifications. The
B16F10 cells were placed in 6-well plates at a concentration of
3  105 cells/ml, and 24 h after plating the cells were treated with
various concentrations of the compounds. After 24 h, the medium
was removed and cells were washed twice with PBS. Later, the cell
pellets containing a known number of cells (usually around
1  106) were dissolved in 1 ml of 1 N NaOH at 60 °C for 30 min
and centrifuged for 10 min at 10,000 rpm. The optical densities
(OD) of the supernatants were measured at 490 nm using an ELISA
reader.
2.5. Cell culture
Human fibroblast were kindly supplied by Surface Science Lab-
oratory of Center for Anti-aging Molecular Science (CAMS) of
Department of Chemistry and School of Molecular Science of Korea
Advanced Institute of Science and Technology (KAIST), maintained
at 37 °C in an incubator with humidified atmosphere of 5% CO2.
Cells were cultured in Dulbecco’s modified Eagle’s medium con-
taining 10% heat-inactivated fetal calf serum, streptomycin
(100 lg/ml), and penicillin (100 unit/ml).
2.6. UV-B irradiation
Cells were exposed to UV-B range at a dose rate of 10–100 mJ/
cm2 (UV Lamp, VL-6LM, Vilber Lourmat, France) and 50 mJ/cm2
dose was identified as the optimum irradiation dose. Therefore,
the 50 mJ/cm2 dose of UV-B was used for further experiments.
2.7. Intracellular reactive oxygen species (ROS) measurement
The DCFH-DA method was used to detect the levels of intracel-
lular ROS (Rosenkranz et al., 1992). DCF-DA diffuses into cells,
where it is hydrolyzed by intracellular esterase to polar 20 ,70 -
dichlorodihydrofluorescein. This non-fluorescent fluorescein ana-
log gets trapped inside the cells, and is oxidized by intracellular
oxidants to a highly fluorescent, 20 ,70 -dichlorofluorescein. The
fibroblast were seeded in 96-well plates at a concentration of
1  105 cells/ml. Sixteen hours after plating, the cells were exposed
to UV-B (50 mJ/cm2) and the compound was treated with various
concentrations. The cells were incubated for an additional 24 h at
37 °C under a humidified atmosphere with 5% CO2. Finally,
DCFH-DA (5 lg/ml) was introduced to the cells, and 20 ,70 -dichloro-
dihydrofluorescein fluorescence was detected at an excitation
wavelength of 485 nm and an emission wavelength of 535 nm,
using a Perkin–Elmer LS-5B spectrofluorometer. In each assay we
used two types of controls; one is negative control (that is non-
irradiated cells) and positive control (that is UV-B irradiated cells).
2.8. Assessment of cell viability
Cell viability was estimated via MTT assay, which is a test of
metabolic competence predicated upon the assessment of mito-
chondrial performance. It is colorimetric assay, which is dependent
on the conversion of yellow tetrazolium bromide to its purple for-
mazan derivative by mitochondrial succinate dehydrogenase in
viable cells (Mosmann, 1983). The fibroblast cells were seeded in
96-well plate at a concentration of 1  105 cells/ml. After 16 h,
the cells were exposed to UV-B (50 mJ/cm2) with the compound
at difference concentrations, and then the cells were incubated
for 24 h at 37 °C. MTT stock solution (2 mg/ml) was then applied
to the wells, to a total reaction volume of 200 ll. After 4 h of the
incubation, the plates were centrifuged for 5 min at 800g, and
 S.-J. Heo et al. / Toxicology in Vitro 23 (2009) 1123–1130
OH
HO OH
Phloroglucinol
HO OH
OH
O
OH
O OH
O
HO
HO
OH
Eckol
Fig. 1. Chemical structures of phlorotannins isolated from E. cava.
the supernatants were aspirated. The formazan crystals in each
well were dissolved in 150 ll of dimethylsulfoxide (DMSO), and
the absorbance was measured via ELISA at the wavelength of
540 nm. Relative cell viability was evaluated in accordance with
the quantity of MTT converted to the insoluble formazan salt.
The optical density of the formazan generated in the control cells
was considered to represent 100% viability. The data are expressed
as mean percentages of the viable cells versus the respective
control.
2.9. Determination of UV-B induced DNA damage by comet assay
Comet assay was performed to determine the irradiation-in-
duced DNA damage (Singh, 2000). The number of cultured cells
was adjusted as 1  105 cells/ml, and the cells were incubated with
the various concentrations of compound for 30 min at 37 °C. After
preincubation, the cells were centrifuged at 300g for 5 min, and
washed using phosphate buffer saline (PBS). Then, the cells were
resuspended in PBS and were exposed to UV-B (50 mJ/cm2). The
non-irradiated control cells were resuspended only in PBS without
UV-B radiation. The cells were washed with 1 ml of PBS and centri-
fuged. The cell suspension was mixed with 75 ll of 0.5% low melt-
ing point agarose (LMPA), and added to the slides precoated with
1.0% normal melting point agarose (NMPA). After keeping them
for 10 min at 4 °C, the slides were covered with another 75 ll of
0.5% LMPA, and kept for 40 min at 4 °C for solidification of the aga-
rose. And the slides were immersed in lysis solution (2.5 M NaCl,
100 mM EDTA, 10 mM Tris, and 1% sodium laurylasarcosine and
1% Triton X-100) for 1 h at 4 °C. The slides were next placed into
an electrophoresis tank containing 300 mM NaOH and 10 mM
Na2EDTA (pH 13.0) for 40 min for DNA unwinding. For electropho-
resis of the DNA, an electric current of 25 V/300 mA was applied for
20 min at 4 °C. The slides were washed three times with a neutral-
izing buffer (0.4 M Tris, pH 7.5) for 5 min at 4 °C, and then treated
with ethanol for another 5 min before staining with 50 ll of ethi-
dium bromide (20 lg/ml). Measurements were made by image
analysis (Kinetic Imaging, Komet 5.0, UK) and fluorescence micro-
scope (LEICA DMLB, Germany), determining the percentage of fluo-
1125
HO OH
O O
O OH
O O
HO OH OH
O
O OH
O
OH
Dieckol
rescence in the tail (tail intensity, TI; 50 cells from each of two
replicate slides).
2.10. Microscopic analysis for dead cells
The type of cell death (apoptosis or necrosis) induced by UV-B
was determined by fluorescent microscopy after staining with
Hoechst 33342 and propidium iodide (PI), as described by Naito
(2001). The fibroblasts were placed in 24-well plates at a concen-
tration of 1  105 cells/ml. Sixteen hours after plating, the cells
were exposed to UV-B (50 mJ/cm2) with the compound at different
concentrations, and then the cells were incubated for 24 h at 37 °C.
After 24 h, 1.5 ll of Hoechst 33342 (10 mg/ml) and PI were added
to each well, step by step, followed by 10 min of incubation at
37 °C. The stained cells were then observed under a fluorescence
microscope equipped with a CoolSNAP-Pro color digital camera,
in order to examine the degree of nuclear condensation.
2.11. Statistical analysis
The data are expressed as the mean ± standard error (S.E.) and
ANOVA test (using SPSS 11.5 statistical software) was used to com-
pare the mean values of each treatment. Significant differences be-
tween the means of parameters were determined by the Duncan
test (p < 0.05).
3. Results
3.1. Inhibitory effect of melanogenesis
The inhibition of tyrosinase-catalyzed dopachrome formation
was evaluated in Fig. 2a. As shown in the figure, dieckol showed
the highest inhibitory effect on tyrosinase as 92.7% and eckol
showed 62.4% at the concentration of 100 lM, but phloroglucinol
did not have tyrosinase inhibitory effect. Especially, dieckol
showed 88.9% of tyrosinase inhibitory activity even at 50 lM,
and the values were higher than that of commercial whitening
agent, kojic acid. To evaluate the inhibitory effect of melanogenesis
1126 S.-J. Heo et al. / Toxicology in Vitro 23 (2009) 1123–1130

300
a 100

Generation of ROS (%)

250 *
* * *
80 *
*
Inhibitory effec of

200 **
tyrosinase (%)

**

60 150
**
100
40
50
20
0
a b 5 50 250
NC PC
0 M of compound + 50 mJ/cm2 UV-B
5 50 100
Concentration ( M) Fig. 3. Effect of phlorotannins isolated from E. cava on UV-B radiation-induced cell
damage. The cells were exposed to UV-B (50 mJ/cm2) and the phlorotannins were
treated with various concentrations (5, 50, and 250 lM). After an incubation of
b 100
24 h, the intracellular ROS was detected by fluorescence spectrophotometer after
DCFH-DA staining. Experiments were performed in triplicate and the data are
Inhibitory effect of melanin

expressed as mean ± SE. Statistical evaluation was performed to compare the

80 experimental groups and positive control groups (UV-B irradiated cells). *p < 0.001,
**p < 0.05. ( ) Phloroglucinol; (Q) eckol; (P) dieckol. aNC: negative control; bPC:
synthesis (%)

60 positive control.

40
120

20
100
Cell viability (%)

**
0 80
** **
10 100 250 **
* *
60 * *
Concentration ( M) *

Fig. 2. Inhibitory effect of phlorotannins isolated from E. cava against melanogen-
esis. (a) Inhibitory effect of phlorotannins isolated from E. cava on mushroom
tyrosinase. L-tyrosine was used as substrate, and kojic acid was used as positive
control. (b) Inhibitory effect of phlorotannins isolated from E. cava on melanin
synthesis. B16F10 mouse melanoma cells were used for this assay and retinol was
used as positive control. Experiments were performed in triplicate and the data are
expressed as mean ± SE. (d) Dieckol; () eckol; (N) kojic acid; (4) phloroglucinol;
(j) retinol.
in cell lines, B16F10 mouse melanoma cells were used for the
determination of inhibitory effect of melanin synthesis. Fig. 2b
illustrated the inhibitory effect of the phloroglucinol, eckol, and
dieckol at different concentrations and recorded as 42.4%, 51.7%,
and 64.9%, respectively at the concentrations of 250 lM. Out of
the phlorotannins, dieckol possess higher inhibitory activity than
those of the other phlorotannins, but the value was relatively lower
than that of the commercial whitening agent, retinol (75.3% at
250 lM).
3.2. Inhibitory effect of ROS generated by UV-B radiation
As shown in Fig. 3, the level of ROS was recorded as 234.1% in
UV-B irradiated cells (positive control) compared to non-irradiated
control cells (negative control). However, the addition of phloro-
tannins to the cells after exposure of UV-B irradiation, intracellular
ROS accumulation was reduced with the increased concentrations
of phlorotannins. Out of those three phlorotannins, dieckol effec-
tively decreased the generated ROS which was recorded as
100.7% at 250 lM. Phloroglucinol and eckol exhibited slightly low-
er activity against UV-B radiation-mediated ROS levels as 192.4%
and 174.4%, respectively at 250 lM.
3.3. Cytotoxicity of phlorotannins after UV-B radiation
The effects of phlorotannins on cell viability in UV-B radiation-
induced fibroblast were measured via MTT assay (Fig. 4). The
40
20
0
a b 5 50 100
NC PC
M of compound + 50 mJ/cm2 UV-B
Fig. 4. Protective effect of phlorotannins isolated from E. cava on UV-B radiation-
induced cell damage. The cells were exposed to UV-B (50 mJ/cm2) and the
phlorotannins were treated with various concentrations (5, 50, and 100 lM). After
an incubation of 24 h, the viability of cells on UV-B radiation was determined by MTT
assay. Experiments were performed in triplicate and the data are expressed as
mean ± SE. Statistical evaluation was performed to compare the experimental
groups and positive control groups (UV-B irradiated cells). *p < 0.001, **p < 0.05. ( )
Phloroglucinol; (Q) eckol; (P) dieckol. aNC: negative control; bPC: positive control.
irradiated cells without phlorotannins (positive control) showed
42.2% cell survival rate, while the viabilities of the cells pre-treated
with phlorotannins before UV-B radiation were increased with the
increased concentrations. Among the tested phlorotannins, dieckol
showed the highest cell survival rate, and recorded the values as
50.5%, 61.4%, and 77.1% at the concentrations of 5, 50, and
100 lM, respectively, compared to non-irradiated cells.
3.4. Protective effects of phlorotannins on UV-B radiation-induced cell
damage
To evaluate whether phlorotannins protect against UV-B radia-
tion, DNA damage induced by UV-B radiation was measured via co-
met assay and was presented as percent fluorescence in damaged
tail (Fig. 5). The UV-B radiation-induced DNA damage in fibroblast
was recorded as 51.5% (positive control), while the negative con-
trol presented as 1.6%. However, the DNA damage was reduced
by the addition of phlorotannins to the cells exposed to the UV-B
radiation. Among the three phlorotannins, dieckol showed higher
 S.-J. Heo et al. / Toxicology in Vitro 23 (2009) 1123–1130 1127

60 20 60 25
a b

Inhibitory effect of cell damage (%)

Inhibitory effect of cell damage (%)

50
Fluorescence in tail (%)

50
16 20

Fluorescence in tail (%)

40 40
12 15

30 30

8 10
20 20

4 5
10 10

0 0 0 0
a b a b
NC PC 5 50 250 NC PC 0.5 5 50

M of compound + 50 mJ/cm2 UV-B M of compound + 50 mJ/cm2 UV-B

60 70
c

Inhibitory effect of cell damage (%)

60
Fluorescence in tail (%)

50

50
40

40
30
30

20
20

10
10

0 0
a b
NC PC 0.5 5 50

M of compound + 50 mJ/cm2 UV-B

Fig. 5. Inhibitory effect of different concentration of phlorotannins isolated from E. cava on UV-B radiation-induced DNA damage using comet assay. The cells were incubated
with various concentrations of phlorotannins. After preincubation, the cells were washed and resuspended using PBS, thereafter, exposed to UV-B (50 mJ/cm2) radiation. The
damaged cells on UV-B radiation was determined by damaged DNA tail movement. Bar graph exhibited % fluorescence in tail and line graph showed the inhibitory effect of
cell damage. Experiments were performed in triplicate and the data are expressed as mean ± SE. Statistical evaluation was performed to compare the experimental groups
and positive control groups (UV-B irradiated cells). *p < 0.001, **p < 0.05. (a) Phloroglucinol; (b) eckol; (c) dieckol. aNC: negative control; bPC: positive control.

protective effect against UV-B radiation-induced cell damage and 4. Discussion

the inhibitory activities were 11.3%, 31.2%, and 57.8% at the con-
centrations of 0.5, 5, and 50 lM, respectively. Fig. 6 illustrated Marine algae have been well-known as an important source to
the DNA migration profiles of the fibroblast cells when treated produce natural bioactive secondary metabolites including phe-
with different concentrations of dieckol are illustrated. The DNA nols and polyphenols with unique linkages (Torres et al., 2008).
was completely damaged and the amount of tail DNA was signifi- E. cava, an edible brown alga with a long history as folk medicine
cantly increased in the group treated with only UV-B radiation in Korea, is abundantly distributed only in Korea and Japan accord-
(Fig. 6b), compared to the negative control (Fig. 6a). However, ing to the latest statistics. The previous reports on E. cava have re-
the amount of tail DNA was significantly decreased with increased vealed that it contains variety of phlorotannin derivates which are
concentrations of dieckol (Figs. 6c–e). Also, we identified morpho- commonly known to have defensive or protective functions against
logical changes of the fibroblast cells induced by UV-B radiation, herbivores (Li et al., 2009). Although some reports suggest that
and the morphology of nuclear chromatin was assessed by staining phlorotannins from algae exhibit antioxidant effect on free radi-
with the combination of two fluorescent dyes, Hoechst 33342 and cals, there are no reports on the effects of the phlorotannins on
PI to detect both apoptosis and necrosis configurations. The micro- melanogenesis and their photo-protective effect on the cell dam-
scopic photograph in Fig. 7 showed that the negative control cells age induced by UV-B radiation, and the underlying mechanism of
had intact nuclei (Fig. 7a), and the UV-B radiation exposed cells phlorotannins. Thus, in this study, the potential whitening effect
(positive control) exhibited significant nuclear fragmentation and of phlorotannins isolated from E. cava was investigated.
destruction which is characteristic of apoptosis (bright blue color) Melanins play a critical role in the absorption of free radicals
and necrosis (red color), respectively (Fig. 7b). However, the and melanogenesis in the skin in a kind of process that produces
amount of fragmentation and destruction of irradiated cells were photo-protective agents against damaging effect from UV. Many
dramatically reduced when the cells were treated with dieckol cosmetic and pharmaceutical companies have tried to find inhibi-
(Figs. 7c–e). tors for melanogenesis. The regulation of cellular pigmentation can
1128 S.-J. Heo et al. / Toxicology in Vitro 23 (2009) 1123–1130

Fig. 6. Photomicrographs of DNA damage and migration observed under dieckol isolated from E. cava. The cells were exposed to UV-B (50 mJ/cm2) and the dieckol was
treated with various concentrations (0.5, 5, and 50 lM). DNA damage and migration were measured by image analysis and fluorescence microscope. (a) Negative control
(non-irradiated cells); (b) positive control (UV-B irradiated cells); (c) cells treated with 0.5 lM dieckol + UV-B radiation at 50 mJ/cm2; (d) cells treated with 5 lM
dieckol + UV-B radiation at 50 mJ/cm2; (e) cells treated with 50 lM dieckol + UV-B radiation at 50 mJ/cm2.

be controlled at the different stages of melanogenesis. Especially,
tyrosinase inhibitors and antioxidants can be used for the inhibi-
tion of cellular pigmentation since the melanin producing process
involved enzymatic and nonenzymatic oxidation reactions (Lee
and Choi, 1999). The proximal pathway of melanogenesis consists
of the enzymatic oxidation of tyrosine or L-DOPA to its correspond-
ing o-dopaquinone, a stepwise catalyzed by tyrosinase (Kim and
Uyama, 2005). After multiple biosynthesis steps, further polymer-
ization yields melanin. In the present study, phlorotannins isolated
from E. cava were examined for their ability to inhibit tyrosinase
activity and to reduce cellular melanin contents in B16F10 cell
lines. From these results, it was deduced that dieckol inhibited cel-
lular pigmentation more effectively than that of the commercial
tyrosinase inhibitor (kojic acid) but significantly lower than that
of melanin synthesis inhibitor (retinol). Moreover, the hexamers
of phloroglucinol (dieckol) had greater activity than those of the
trimer (eckol) and monomer (phloroglucinol). Park and Chang
(1997) reported that the tyrosinase inhibitory activities of the phe-
nolic compounds were in the sequence of trimer, dimmer, and
monomer, which is in agreement with the findings of the present
study. Therefore, dieckol from E. cava presented strong tyrosinase
inhibitory and melanin synthesis inhibitory activities. Hence, diec-
kol can be applied as a natural whitening material.
Oxidative stress may be induced by increasing generation of
ROS and other free radicals. UV radiation can induce the formation
of ROS such as singlet oxygen and superoxide anion in the skin,
promoting biological damage in exposed tissues via iron-catalyzed
oxidative reactions (Wang et al., 2006). Generation of intracellular
ROS can be detected using oxidation sensitive dye DCFH-DA as the
substrate. Fluorescent probes have been widely employed to mon-
itor oxidative activity in cells. During labeling, non-fluorescent
DCFH-DA dye that freely penetrates into the cells gets hydrolyzed
by intracellular esterase to DCFH, and traps inside the cells (Veer-
man et al., 2004). Therefore, an increase in the cellular fluorescence
level reveals the elevated levels of ROS. Many types of death stim-
uli induce the increasing cellular levels of ROS, and the elimination
of the induced ROS through the use of antioxidants protects the
cells from those stimuli. Therefore, ROS are believed to act as key
mediators of cell death (Martindale and Holbrook, 2002; Kim
et al., 2008). These results suggest that dieckol can be developed
into a potential bio-molecular candidate to inhibit ROS formation.
As phlorotannins were found to exert a ROS scavenging effect, it
was further evaluated with regard to its protective effects against
UV-B radiation-induced cell damage. The effects of phlorotannins
on cell viability in UV-B radiation-induced fibroblast were mea-
sured via MTT assay. MTT conversion could provide an indirect
indicator of cell metabolism, since MTT reduction to formazan in
viable cells take place via reactions catalyzed by mitochondrial
dehydrogenases, coupled to oxidative phosphorylation (Ekmekcio-
glu et al., 1999). Our results showed that formazan was reduced
due to UV-B radiation, however, significantly increased with the
addition of phlorotannins, especially in dieckol. Phlorotannins, well
known marine algal polyphenols, are recognized to have defensive
or protective functions against oxidative stress (Kang et al., 2003).
Although some reports suggest that the phlorotannins from algae
exhibit antioxidant effect on free radicals and H2O2, there are no
reports on the protective effects against UV-B radiation of dieckol.
These results demonstrated that dieckol possess the potential
 S.-J. Heo et al. / Toxicology in Vitro 23 (2009) 1123–1130 1129

Fig. 7. Protective effect of dieckol isolated from E. cava on UV-B radiation-induced cell damage. The cells were exposed to UV-B (50 mJ/cm2) and the dieckol was treated with
various concentrations (0.5, 5, and 50 lM). After an incubation of 24 h, cellular morphological changes were observed under a fluorescence microscope after Hoechst 33342
and PI double staining. (a) Negative control (non-irradiated cells); (b) positive control (UV-B irradiated cells); (c) cells treated with 0.5 lM dieckol + UV-B radiation at 50 mJ/
cm2; (d) cells treated with 5 lM dieckol + UV-B radiation at 50 mJ/cm2; (e) cells treated with 50 lM dieckol + UV-B radiation at 50 mJ/cm2.

inhibitory effects on intracellular oxidative damage induced by
UV-B radiation, and the antioxidant activities were associated with
the improvement of the cell viability as compared to our previous
study (Ahn et al., 2007).
The skin possesses an elaborate antioxidant defense system to
protect it from oxidative stress, but excessive exposure to reactive
oxygen species can shift the prooxidant–antioxidant balance of the
skin toward the more oxidative state. The resulting oxidative stress
causes many adverse effects and pathological conditions (Anan-
thaswamy and Kanjilal, 1996; Finkel and Holbrook, 2000). Under
these circumstances, regular intake of dietary antioxidants or
treatment of the skin with products containing UV protective
ingredients may be a useful strategy for preventing UV-B induced
damage. Therefore, in this study, we report the protective effect
of phlorotannins as antioxidant and UV protective agent for UV-B
radiation-induced cell damage via comet assay and morphological
analysis. It was clearly demonstrated that dieckol completely re-
duced both DNA damage and the nuclear fragmentation induced
by UV-B radiation. Several natural compounds have gained consid-
erable attention as skin photo-protective agents. Plants can change
their phenolic metabolisms under UV-B radiation, which supply
enough phenolic compounds to screen UV-B light, and sometimes
these molecules might function as antioxidant to quench ROS gen-
erated by UV-B radiation (Booij-James et al., 2000; Sudheer et al.,
2007; Skandrani et al., 2009). Phlorotannins are well known poly-
phenols in marine algae, which have various biological activities
including antioxidant, antitumor, antihypertension, and anti-
inflammatory effects. Among them, antioxidant activity related to
UV protection is intensively focused due to the currently growing
demand from the cosmeceutical industry where they are inter-
ested in anti-aging and whitening natural products. Dieckol, phe-
nolic moiety, is a kind of phlorotannin which consists of six
phloroglucinol units and functional hydroxyl groups. Recent stud-
ies have demonstrated that the protective effect against oxidative
stress induced by ROS and UV radiation is correlated with the
number and position of hydrogen- donating hydroxyl groups on
the aromatic ring of the phonolic molecules, and is also affected
by other factors, such as other H-donating groups (–NH, –SH), etc
(Rice-Evans et al., 1995; Lien et al., 1999). Our results indicated
that dieckol has more functional hydroxyl groups than other tested
phlorotannins, therefore, the dieckol can easily be applied in anti-
oxidant and cosmeceutical industries as a natural compound from
marine biomass.
5. Conclusion
In the present study, we isolated marine algal polyphenol,
phlorotannins from E. cava including phloroglucinol, eckol, and
dieckol, and their effects on melanogenesis were evaluated via
the inhibitory effect of tyrosinase and melanin synthesis. Also,
the protective effects of the compounds against photo-oxidative
stress induced by UV-B radiation were evaluated via DCFH-DA,
MTT, comet assay, and microscopic analysis. Among the isolated
phlorotannins, dieckol showed prominent inhibitory activity
against melanogenesis and effectively reduced UV-B radiation-in-
duced cell damages. These results indicated that dieckol can be
used as an effective natural source to make cosmeceutical or phar-
maceutical products.
Acknowledgements
This research was supported by a grant (M-2008-01) from Mar-
ine Bioprocess Research Center of the Marine Bio 21 Center funded
by the Ministry of Land, Transport and Maritime, Republic of Korea.
1130 S.-J. Heo et al. / Toxicology in Vitro 23 (2009) 1123–1130

References Nagayama, K., Iwamura, Y., Shibata, T., Hirayama, I., Nakamura, T., 2002. Bactericidal
activity of phlorotannins from the brown alga Ecklonia kurome. Journal of
Antimicrobial Chemotherapy 50, 889–893.
Ahn, G.N., Kim, K.N., Cha, S.H., Song, C.B., Lee, J., Heo, M.S., Yeo, I.K., Lee, N.H., Lee,
Naito, Y., 2001. Neutrophil-dependent oxidative stress in gastrointestinal
Y.H., Kim, J.S., Heu, M.S., Jeon, Y.J., 2007. Antioxidant activities of phlorotannins
inflammation. In: Yoshikawa, T. (Ed.), Oxidative Stress and Digestive Disease.
purified from Ecklonia cava on free radical scavenging using ESR and H2O2-
Kager Basel, pp. 24–40.
mediated DNA damage. European Food Research and Technology 226, 71–79.
Nakamura, T., Nagayama, K., Uchida, K., Tanaka, R., 1996. Antioxidant activity of
Ananthaswamy, H.N., Kanjilal, S., 1996. Oncogenes and tumor suppressor genes in
phlorotannins isolated from the brown alga Eisenia bicyclis. Fisheries Science 62,
photocarcinogenesis. Photochemistry and Photobiology 63, 428–432.
923–926.
Booij-James, I.S., Dube, S.K., Jansen, M.A.K., Edelman, M., Mattoo, A.K., 2000.
Nakayama, T., Takahashi, M., Fukuyama, Y., Kinzyo, Z., 1989. An anti-plasmin
Ultraviolet-B radiation impacts light-mediated turnover of the photosystem II
inhibitor, eckol, isolated from the brown alga Ecklonia kurome OKAMURA.
reaction center heterodimer in Arabidopsis mutants altered in phenolic
Agricultural and Biological Chemistry 63, 3025–3030.
metabolism. Plant Physiology 124, 1275–1283.
Park, Y.H., Chang, S.K., 1997. Screening of inhibitory effect of edible mushroom on
Caddeo, C., Teskač, K., Sinico, C., Kristl, J., 2008. Effect of resveratrol incorporated in
tyrosinase and isolation of active component. Journal of Food Hygiene and
liposomes on proliferation and UV-B protection of cells. International Journal of
Safety 12, 195–199.
Pharmaceutics 363, 183–191.
Rice-Evans, C.A., Muller, N.J., Bolwell, P.G., BramLey, P.M., Pridham, J.B., 1995. The
Dykens, J.A., Shick, J.M., Benoit, C., Buettner, G.R., Winston, G.W., 1992. Oxygen
relative antioxidant activities of plant-derived polyphenolic flavonoids. Free
radical production in the sea anemone Anthopleura elegantissima and its
Radical Research 22, 375–383.
endosymbiotic algae. Journal of Experimental Biology 168, 219–241.
Rosenkranz, A.R., Schmaldienst, S., Stuhlmeier, K.M., Chen, W., Knapp, W., Zlabinger,
Ekmekcioglu, E., Strauss-Blasche, G., Leibetseder, V.J., Marktl, W., 1999.
G.J., 1992. A microplate assay for the detection of oxidative products using 20 ,70 -
Toxicological and biochemical effects of different beverages on human
dichlorefluorescein-diacetate. Journal of Immunological Methods 156, 39–45.
intestinal cells. Food Research International 32, 421–427.
Sabry, O.M.N., Andrews, S., McPhail, K.L., Geoger, D.E., Yokochi, A., LePag, K.T.,
Finkel, T., Holbrook, N.J., 2000. Oxidants, oxidative stress and the biology of ageing.
Murray, T.F., Gerwick, W.H., 2005. Neurotoxic meoditerpenoids from the
Nature 408, 239–247.
tropical marine brown alga Stypopodium flabelliorme. Journal of Natural
Fuchs, J., 1998. Potentials and limitations of the natural antioxidants RRR-alpha-
Product 68, 1022–1030.
tocopherol, L-ascorbic acid and b-carotene in cutaneous photoprotection. Free
Singh, N.P., 2000. Microgels for estimation of DNA strand breaks, DNA protein cross
Radical Biology and Medicine 25, 848–873.
links and apoptosis. Mutation Research 455, 111–127.
Heo, S.J., Jeon, Y.J., 2008. Radical scavenging capacity and cytoprotective effect of
Skandrani, I., Bouhlel, I., Limem, I., Boubaker, J., Bhouri, W., Neffati, A., Sghaier, M.B.,
enzymatic digests of Ishige okamurae. Journal of Applied Phycology 20, 1087–
Kilani, S., Ghedira, K., Ghedira-Chekir, L., 2009. Moricandia arvensis extracts
1095.
protect against DNA damage, mutagenesis in bacteria system and scavenge the
Heo, S.J., Park, E.J., Lee, K.W., Jeon, Y.J., 2005. Antioxidant activities of enzymatic
superoxide anion. Toxicology In Vitro 23, 166–175.
extracts from brown seaweeds. Bioresource Technology 96, 1613–1623.
Sturm, R.A., Teasdale, R.D., Box, N.F., 2001. Human pigmentation genes:
Jimenez-Escrig, A., Jimenez-Jimenez, I., Pulido, R., Saura-Calixto, F., 2001.
identification, structure and consequences of polymorphic variation. Gene
Antioxidant activity of fresh and processed edible seaweeds. Journal of the
277, 49–62.
Science of Food and Agriculture 81, 530–534.
Sudheer, A.R., Muthukumaran, S., Devipriya, N., Menon, V.P., 2007. Ellagic acid, a
Kajiwara, T., Matsui, K., Akakabe, Y., Murakawa, T., Arai, C., 2006. Antimicrobial
natural polyphenol protects rat peripheral blood lympocytes against nicotine-
browning-inhibitory effect flavor compounds in seaweeds. Journal of Applied
induced cellular and DNA damage in vitro: with the comparison of N-
Phycology 18, 413–422.
acetylcysteine. Toxicology 230, 11–21.
Kang, K., Park, Y., Hwang, H.J., Kim, S.H., Lee, J.G., Shin, H.C., 2003. Antioxidative
Sugiura, Y., Matsuda, K., Yamada, Y., Nishikawa, M., Shioya, K., Katsuzaki, H., Imai, K.,
properties of brown algae polyphenolics and their perspectives as
Amano, H., 2006. Isolation of a new anti-allergic phlorotannin,
chemopreventive agents against vascular risk factors. Archives of Pharmacal
phlorofucofuroeckol-B, from an edible brown alga, Eisenia arborea. Bioscience
Research 26, 286–293.
Biotechnology and Biochemistry 70, 2807–2811.
Kim, E.M., Yang, H.S., Kang, S.W., Ho, J.N., Lee, S.B., Um, H.D., 2008. Amplification of
Tanaka, K., Hasegawa, J., Asamitsu, K., Okamoto, T., 2007. Magnolia ovovata extract
the c-irradiation-induced cell death pathway by reactive oxygen species in
and its active component magnolol prevent skin photoaging via inhibition of
human U937 cells. Cellular Signalling 20, 916–924.
nuclear factor jB. European Journal of Pharmacology 565, 212–219.
Kim, Y.J., Uyama, H., 2005. Tyrosinase inhibitors from natural and synthetic sources:
Tengamnuay, P., Pengrungruangwong, K., Pheansri, I., Likhitwitayawuid, K., 2006.
structure, inhibition mechanism and perspective for the future. Cellular and
Artocarpus lakoocha heartwood extract as a novel cosmetic ingredient:
Molecular Life Science 62, 1707–1723.
evaluation of the in vitro anti-tyrosinase and in vivo skin whitening activities.
Lee, K.K., Choi, J.D., 1999. The effects of Areca catechu L extract on anti-inflammation
International Journal of Cosmetic Science 28, 269–276.
and anti-melanogenesis. International Journal of Cosmetic Science 21, 275–284.
Thiele, J.J., Podda, M., Packer, L., 1997. Tropospheric ozone an emerging
Li, Y., Qian, Z.J., Ryu, B., Lee, S.H., Kim, M.M., Kim, S.K., 2009. Chemical components
environmental stress to skin. Biological Chemistry 378, 1299–1305.
and its antioxidant properties in vitro: an edible marine brown alga, Ecklonia
Torres, M.A., Barros, M.P., Campos, S.C., Pinto, E., Rajamani, S., Sayre, R.T., Colepicolo,
cava. Bioorganic and Medicinal Chemistry 17, 1963–1973.
P., 2008. Biochemical biomarkers in algae and marine pollution: a review.
Lien, E.J., Ren, S.J., Bui, H.Y.H., Wang, R.B., 1999. Quantitative structure–activity
Ecotoxicology and Environmental Safety 71, 1–15.
relationship analysis of phenolic antioxidants. Free Radical Biology and
Tsuboi, T., Kondoh, H., Hiratsuka, J., Mishima, Y., 1998. Enhanced melanogenesis
Medicine 26, 285–294.
induced by tyrosinase gene-transfer increases boron-uptake and killing effect of
Longstreth, J., de Gruijl, F.R., Kripke, M.L., Abseck, S., Arnold, F., Slaper, H.I., Velders,
boron neutron capture therapy for amelanotic melanoma. Pigment Cell
G., Takizawa, Y., van der Leun, J.C., 1998. Health risks. Journal of Photochemistry
Research 11, 275–282.
and Photobiology B: Biology 46, 20–39.
Vanni, A., Gastaldi, D., Giunata, G., 1990. Kinetic investigations on the double
Marrot, L., Belaidi, J.P., Chaubo, C., Meunier, J.R., Perez, P., Agapakis-Causse, C., 2001.
enzymatic activity of the tyrosinase mushroom. Annali di Chimica 80, 35–60.
Fluoroquinolones as chemical tools to define a strategy for photogenotoxicity
Veerman, E.C., Nazmi, K., Hof, W.V., Bolscher, J.G., Hertog, A.L.D., Amerongen, A.V.N.,
in vitro assessment. Toxicology In Vitro 15, 131–142.
2004. Reactive oxygen species play no role in the candidacidal activity of the
Martindale, J.L., Holbrook, N.J., 2002. Cellular response to oxidative stress: signaling
salivary antimicrobial peptide histatin 5. Biochemical Journal 381, 447–452.
for suicide and survival. Journal of Cellular Physiology 192, 1–15.
Wang, K.H., Lin, R.D., Hsu, F.L., Huang, Y.H., Chang, H.C., Huang, C.Y., Lee, M.H., 2006.
Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival:
Cosmetic applications of selected traditional Chinese herbal medicines. Journal
application to proliferation and cytotoxicity assays. Journal of Immunological
of Ethnopharmacology 106, 353–359.
Methods 65, 55–63.