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Botanica Marina 52 (2009): 268–277 2009 by Walter de Gruyter • Berlin • New York. DOI 10.1515/BOT.2009.037
Article in press - uncorrected proof
2009/70
Antioxidant and cytotoxic activities of some red algae
(Rhodophyta) from Brittany coasts (France)
Mayalen Zubia*, Marie-Sophie Fabre,
Ve´ ronique Kerjean and Eric Deslandes
Plateforme BIODIMAR/UBO, Universite´ de Bretagne
Occidentale, 305 rue Claude Shannon, 29280 Plouzane´,
France, e-mail: mayalen_zubia@yahoo.fr
* Corresponding author
Abstract
We assessed the antioxidant activity of crude extracts
from 24 rhodophyte species from Brittany coasts using
three complementary methods (2,2-diphenyl-1-picryhy-
drazyl, reducing activity, and b-carotene-linoleic acid
system). We also examined phenolic contents. Cytotoxic
activities were determined with three different cancer cell
lines. Four species (Aglaothamnion pseudobyssoides,
Furcellaria fastigiata, Polysiphonia lanosa, and Heterosi-
phonia plumosa) had high antioxidant activity and high
phenolic content. The extract from Brongniartella bys-
soides had the highest antioxidant potential, which was
also found to be equivalent to the antioxidant activities
of some commercial antioxidants. In the b-carotene sys-
tem, extracts from Porphyra leucosticta and Porphyra
purpurea had some specific antioxidant activity. Further-
more, Asparagopsis armata, B. byssoides and H. plumosa
extracts had strong cytotoxic activities against Daudi and
Jurkat cells.
Keywords: antioxidant activity; cytotoxic activity;
macroalgae.
Introduction
In Asian countries, macroalgae are traditionally traded as
food items, e.g., sushi wrappings, seasonings, condi-
ments, and vegetables, and for the phycocolloid industry.
In recent years, the market for macroalgae has consid-
erably expanded into the pharmaceutical and para-phar-
maceutical sectors owing of their exceptional richness in
bioactive compounds (e.g., antimicrobial, anti-inflamma-
tory, and antitumoral activities) (Smit 2004, Kornprobst
2005). There is a huge potential for macroalgae in the
food, medical, and cosmetic industries, especially red
macroalgae that form a very heterogeneous group with
regard to their chemical composition (Kor nprobst 2005).
Among the most relevant compounds found in algae,
antioxidants are probably those that have attracted most
interest. Antioxidants are considered key compounds in
the fight against various diseases (e.g., cancer, chronic
inflammation, atherosclerosis, cardiovascular disorders)
and the aging process (Kohen and Nyska 2002). More-
over, the interest in employing antioxidants from natural
sources has been considerably enhanced by consumers’
preferences for natural products and concerns about the
toxic effects of synthetic antioxidants (Ito et al. 1986).
Algae, as photosynthetic organisms, are exposed to a
combination of light and high oxygen concentrations,
which induces the formation of free radicals and other
oxidative reagents. The absence of structural damage in
the algae implies that these organisms are able to gen-
erate antioxidants to protect themselves against oxida-
tion. In this regard, macroalgae are considered to be rich
in natural antioxidants (e.g., phlorotannins, ascorbic acid,
tocopherols, carotenoids) (Plaza et al. 2008).
Another area that has been the focus of much attention
is the search for anticancer drugs, as marine molecules
have shown promising results for different stages of can-
cer development (Mayer and Gustafson 2006). Among
marine organisms, numerous macroalgae have potent
cytotoxic activities (see reviews in Gu¨ ven et al. 1990,
Smit 2004, Mayer and Gustafson 2006) and algal con-
sumption has been suggested as a chemo-preventive
agent against several cancers (Funahashi et al. 2001,
Yuan and Walsh 2006). Halogenated terpenoids from red
algae are considered to be promising secondary metab-
olites in anticancer research. Indeed, dehydrothyrsiferol
and halomon, extracted from Laurencia viridis (Pec et al.
2003) and Portieria hornemanii (Egorin et al. 1997),
respectively, have been developed to the preclinical
phase.
The coasts of Brittany (France) are characterized by
great macroalgal species richness (around 700 species;
Dizerbo and Herpe´ 2003). Among them, only few, mainly
brown algae, have been studied for their antioxidant
capacities (Cerantola et al. 2006, Connan et al. 2006,
2007), their anti-fouling (Hellio et al. 2000, 2001, 2004),
and antitumoral (Moreau et al. 2006) activities.
This study aimed to assess the antioxidative and cyto-
toxic capabilities of 24 red algae growing along the
shorelines of Brittany. To gain more insight into antioxi-
dant processes, the antioxidative activities of the crude
extracts were characterized by three biochemical meth-
ods: 2,2-diphenyl-1-picryhydrazyl (DPPH) radical-scav-
enging activity, reducing activity, and b-carotene-linoleic
acid system. The total phenolic contents of these
extracts were also examined, and their cytotoxic activi-
ties were determined with three different cancer cell lines
(Daudi, Jurkat, and K562).
Materials and methods
Collection
Samples of 24 species of red macroalgae were collected
along the coasts of Brittany between 2006 and 2007
M. Zubia et al.: Biological activities of some red algae 269
Article in press - uncorrected proof
Table 1 Information on the red macroalgae studied.
Number Species Order Family Place of Date of
collection collection
1 Aglaothamnion pseudobyssoides (P.L. Crouan et Ceramiales Ceramiaceae Callot June 2007
H.M. Crouan) Halos
2 Asparagopsis armata Harvey Bonnemaisoniales Bonnemaisoniaceae Anse de Melon March 2007
3 Brongniartella byssoides (Goodenough et Ceramiales Rhodomelaceae Callot June 2007
Woodward) F. Schmitz
4 Calliblepharis jubata (Goodenough et Woodward) Gigartinales Cystocloniaceae Anse de Melon March 2007
Ku¨ tzing
5 Callithamnion tetragonum (Withering) S.F. Gray Ceramiales Ceramiaceae Anse de Melon May 2007
6 Callophyllis laciniata (Hudson) Ku¨ tzing Gigartinales Kallymeniaceae Anse de Melon May 2007
7 Caulacanthus ustulatus (Mertens ex Turner) Gigartinales Caulacanthaceae Callot June 2007
Ku¨ tzing
8 Ceramium ciliatum (J. Ellis) Ducluzeau Ceramiales Ceramiaceae Anse de Melon May 2007
9 Cystoclonium purpureum (Hudson) Batters Gigartinales Cystocloniaceae Anse de Melon May 2007
10 Dilsea carnosa (Schmidel) Kuntze Gigartinales Dumontiaceae Bertheaume July 2007
11 Dumontia contorta (S.G. Gmelin) Ruprecht Gigartinales Dumontiaceae Callot March 2006
12 Furcellaria fastigiata (Turner) J.V. Lamouroux Gigartinales Furcellariaceae Anse de Melon March 2007
13 Gastroclonium ovatum (Hudson) Papenfuss Rhodymeniales Champiaceae Anse de Melon April 2007
14 Gracilaria gracilis (Stackhouse) M. Steentoft, Gracilariales Gracilariaceae Callot June 2007
L.M. Irvine et W.F. Farnham
15 Grateloupia filicina (J.V. Lamouroux) C. Agardh Halymeniales Halymeniaceae Callot June 2007
16 Heterosiphonia plumosa (J. Ellis) Batters Ceramiales Dasyaceae Anse de Melon May 2007
17 Kallymenia reniformis (Turner) J. Agardh Gigartinales Kallymeniaceae Porz Liogan June 2007
18 Lomentaria articulata (Hudson) Lyngbye Rhodymeniales Lomentariaceae Anse de Melon March 2007
19 Lomentaria clavellosa (Turner) Gaillon Rhodymeniales Lomentariaceae Anse de Melon May 2007
20 Plocamium cartilagineum (Linnaeus) P.S. Dixon Plocamiales Plocamiaceae Anse de Melon May 2007
21 Polyneura bonnemaisonii (C. Agardh) Ceramiales Delesseriaceae Anse de Melon March 2007
Maggs et Hommersand
22 Polysiphonia lanosa (Linnaeus) Tandy Ceramiales Rhodomelaceae Anse de Melon May 2007
23 Porphyra leucosticta Thuret Bangiales Bangiaceae Anse de Melon April 2007
24 Porphyra purpurea (Roth) C. Agardh Bangiales Bangiaceae I
ˆ
le de Bec July 2007
(Table 1). Once harvested, they were stored in plastic
bags for transport to the laboratory. Voucher specimens
of all species were pressed and stored in 4% formalin
for identification (Cabioc’h et al. 2006). All of the samples
were washed thoroughly with freshwater to remove salts,
sand, and epiphytes, and then stored at -208C. Each
macroalgal sample was lyophilized and pulverized into
powder before extraction.
Preparation of crude algal extracts
Extraction was carried out with an Accelerated Solvent
Extraction system (ASE 300) equipped with a solvent
controller unit (Dionex, Yvelines, France). A preliminary
study was carried out to improve the extraction yields by
optimizing parameters (unpublished data). The red algal
extractions performed in this study were carried out with
the optimized parameters as follows: 10 g of lyophilized
samples were mixed with 10 g of Fontainebleau sand as
a dispersing agent, and placed in a 66-ml stainless steel
extraction cell equipped at its outlet with a cellulose filter.
All extractions were performed with dichloromethane
methanol (1:1, v:v) at 758C and 1500 psi (103.42 bar)
during two static 7-min cycles. Each sample was extract-
ed twice, and then the cell was rinsed with solvent and
purged with a flow of nitrogen. The extracts were then
filtered through a grade-4 Whatman filter and concen-
trated to 10 ml under reduced pressure prior to storage
at -208C.
Antioxidant assays
DPPH radical-scavenging activity 2,2-Diphenyl-1-picry-
hydrazyl radical (DPPH) radical-scavenging activity was
determined using the method developed by Brand-Wil-
liams et al. (1995) and modified by Fukumoto and Mazza
(2000). The fundamental principle of the DPPH method
is the reduction of the DPPH radical in alcoholic solution
by H-donor antioxidant (AH) to form the non-radical form
DPPH-H. In a 96-well microplate, 22
ml of sample were
mixed with 200
ml of the DPPH solution (25 mg l
-1
)pre-
pared daily. Samples were prepared in triplicate and at
least seven different concentrations (0–40 mg ml
-1
) were
used for each extract. The DPPH solution and the sam-
ples were prepared with 80% methanol. Due to the color
of the extracts, blanks had to be prepared by mixing
22
ml of sample with 200 ml of 80% methanol. The reac-
tion was incubated for 2 h in the dark at room temper-
ature, and then the absorbance was read at 515 nm with
a multi-well spectrophotometer (Sunrise, TECAN, Lyon,
France). The DPPH concentration in the reaction medium
was calculated from a calibration curve (ns8, rs0.99)
determined by the following linear regression:
wDPPHxs(Abs–0.0398)/0.0137 to further deduce the per-
centage of remaining DPPH (% DPPH). A curve of extract
concentration against % DPPH was generated to esti-
mate the concentration of extract needed to cause a
50% reduction of the initial DPPH concentration. This
value is known as EC
50
(efficient concentration when
50% oxidation is achieved, also called oxidation index)
270 M. Zubia et al.: Biological activities of some red algae
Article in press - uncorrected proof
and was expressed in units of mg ml
-1
. This assay was
carried out in triplicate for each sample, and the mean
values were used to calculate the EC
50
. Ascorbic acid, a-
tocopherol, butylated hydroxyanisole (BHA) and butylat-
ed hydroxytoluene (BHT) were used as positive controls.
Reducing activity The method of Kuda et al. (2005)
was used, after slight modifications (incubation time and
wavelength), to assess the reducing activity of each
extract. This method relies on the evaluation of total anti-
oxidant capacity of a given extract from the redox poten-
tials of the compounds. Aliquots of extracts (0.2 ml) at
four different concentrations (50, 100, 200, and 500
mg l
-1
) were mixed with phosphate buffer (0.2 ml, 0.2
M
,
pH 7.2) and potassium ferricyanide wK
3
Fe(CN)
6
x (0.2 ml,
1%). After incubation at 508C for 30 min, the mixture was
cooled down prior to the addition of 0.2 ml of trichloro-
acetic acid (10%). Then, 125-
ml aliquots of this mixture
were transferred to a 96-well microplate before addition
of 20
ml of 0.1% FeCl
3
=6H
2
O to each well. Increased
absorbance of the reaction mixture at 620 nm indicated
increased reducing activity. Absorbance was read with a
multi-well spectrophotometer (Sunrise, TECAN) and
then transformed into a percentage of inhibition in com-
parison to a blank (ethanol). This assay was carried out
in triplicate for each sample and positive controls (ascor-
bic acid, a-tocopherol, BHA and BHT).
b-Carotene-linoleic acid system The antioxidant
activities of samples assayed by the b-carotene-linoleic
acid system were measured according to the method
developed by Koleva et al. (2002) (emulsion preparation)
and by Zhang et al. (2007). This method is based on the
loss of the yellow color from b-carotene due to its reac-
tion with the radicals formed by linoleic acid oxidation;
b-carotene bleaching is, thus, slowed down in the pres-
ence of antioxidants. After dissolution of b-carotene
(1 mg) in 5 ml of chloroform, 1 ml of this solution was
mixed with linoleic acid (25
ml) and Tween-40 (200 mg).
After removal of chloroform by evaporation at 408C under
vacuum, 50 ml of distilled water oxygenated by air-bub-
bling were added slowly to the semi-solid residue under
vigorous stirring to form an emulsion. A 96-well micro-
plate was loaded with 50
ml per well of the samples or
positive controls (a-tocopherol, BHA and BHT prepared
in ethanol) and 200
ml of the emulsion. Four final con-
centrations were tested (50, 100, 200, and 500 mg l
-1
)
and ethanol was used as blank. All determinations were
carried out in triplicate. The absorbance value at 450 nm
was read with a multi-well spectrophotometer (Sunrise,
TECAN) with addition of the emulsion considered as the
starting time of the reaction (ts0 min). After covering the
plate with a film (Seal Plate, VWR, Fontenay sur Bois,
France) and incubation at 508C for 3 h, the absorbance
was measured every 30 min. The antioxidant activity (AA)
of the extracts was evaluated as the percentage inhibi-
tion of bleaching of b-carotene using the following for-
mula: AAsw 1–(A
0
–A
t
)/(A9
0
–A9
t
)x=100, where A
0
is the
absorbance of the sample at ts0 min and A9
0
is the
absorbance of the control at ts0 min, A
t
and A9
t
are
absorbances of the sample and control at ts3h.
Total phenolic content
The total phenolic content of algal extracts was deter-
mined by spectrophotometry using the Folin-Ciocalteu
method from Connan (2004). The samples (100
ml) were
mixed with 50
ml Folin-Ciocalteu reagent, 200 ml of 20%
sodium carbonate solution, and 650
ml of distilled water.
The mixture was incubated at 708C in the dark for 10 min.
After production of a blue color, absorbance was read at
700 nm. The total content of phenolic compounds was
expressed as % of dry weight (% dw) based on a stan-
dard curve of phloroglucinol. This analysis was carried
out in triplicate for each extract.
Cell culture
Daudi (Human Burkitt’s lymphoma), Jurkat (Human leu-
kemic T-cell lymphoblast), and K562 (Human chronic
myelogenous leukemia) cells were obtained from ECACC
(European Collection of Cell Cultures, Salisbury, UK) and
grown in RPMI 1640 medium with
L
-glutamine (Lonza,
Basel, Switzerland) supplemented with 10% of heat inac-
tivated fetal bovine serum (FBS; Cambrex Bioscience,
Saint-Beauzire, France), and 20% for Daudi cells. Cells
were plated at 5=10
4
cells in 200 ml per well onto 96-
flat-bottomed well ELISA plates and incubated at 378C
in 5% CO
2
for 24 h. Blanks (medium without cells) were
prepared under the same conditions. Seaweed extracts
were added to the cells at a concentration of 100
mgml
-1
.
Dimethyl sulfoxide (DMSO) was used as control and was
added to the cells at a concentration of 100
mgml
-1
.
Seaweed extracts, blank, and control were prepared in
triplicate. Subsequently, the plates were incubated for
24 h.
Cytotoxic assay
The cytotoxic activities of these crude extracts were
investigated by a cytotoxic assay adapted from the
method by Ishiyama et al. (1995). To assess mitochon-
drial function, mitochondrial dehydrogenase (succinate-
tetrazolium-reductase) activity was determined with the
WST-1 (4-w3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetra-
zoliox-1,3-benzene disulfonate) colorimetric assay (Roche
Diagnostics, Meylan, France); WST-1 is a tetrazolium dye
containing an electron coupling reagent which is cleaved
by the mitochondrial dehydrogenase enzyme to a water-
soluble formazan dye, and there is a direct correlation
between this reaction and the number of metabolically
active cells. After 24 h of incubation, 10
ml of the for-
mazan dye were added to each well prior to a 3-h incu-
bation at 378C. Absorbance was measured in triplicate at
450 nm with a multi-well spectrophotometer (Infinite,
TECAN) in blank, control, and tested cells (with sea-
weeds extracts).
Cells were plated a second time and the experiment
was repeated. The results were then expressed in per-
centage of viability in comparison to the control.
Statistical analysis
All statistical analyses were performed with Statistica 6
software (StatSoft Inc., Maison-Alfort, France). The data
were tested for normality (Shapiro-Wilk’s test) and sub-
M. Zubia et al.: Biological activities of some red algae 271
Article in press - uncorrected proof
Figure 1 DPPH radical-scavenging activities expressed as the oxidation index EC
50
(mg ml
-1
, mean
"
SD, ns3) of red algal crude
extracts and controls (ascorbic acid, a-tocopherol, BHA and BHT).
Significant differences between radical-scavenging activities of the crude extracts and the controls were determined by the Tukey
HSD test (p-0.05) and are indicated by different letters (a–u).
jected to the Bartlett test to verify the homogeneity of
variance. One-way analysis of variance was carried out
to compare antioxidant activities between extracts, after
transformation of data (arcsin or log) to obtain homoge-
neity of variance when required. A post-hoc test (Tukey
HSD) was carried out when data showed significant dif-
ferences (p-0.05) to classify antioxidant activity means
measured for extracts and positive controls. Student’s
t-test was used to compare cytotoxic activity of the
extracts with the control (p-0.01).
The Pearson correlation coefficient (r) was also calcu-
lated (p-0.05) to evaluate the relationship between the
different methods used to measure antioxidant activities
of the extracts and to determine whether phenolic com-
pounds were implicated in the antioxidant capacity and
in the cytotoxic activities.
Results
Antioxidant activity
Figure 1, Tables 2 and 3 report main results of the
assessment by DPPH radical-scavenging-activity, reduc-
ing activity, and the b-carotene-linoleic acid system,
respectively, of the crude extract antioxidative activities.
All the species collected for this study had DPPH rad-
ical-scavenging activities (Figure 1). The crude extract
from Brongniartella byssoides had the lowest EC
50
value
(0.14
"
0.01 mg ml
-1
), which was similar to the DPPH rad-
ical-scavenging activity of a-tocopherol (0.14
"
0.01 mg
ml
-1
). Furcellaria fastigiata (1.39
"
0.04 mg ml
-1
), Polysi-
phonia lanosa (2.71
"
0.01 mg ml
-1
), Aglaothamnion pseu-
dobyssoides (3.01
"
0.05 mg ml
-1
), and Heterosiphonia
272 M. Zubia et al.: Biological activities of some red algae
Article in press - uncorrected proof
Table 2 Reducing activity of red algal crude extracts at different concentrations (50, 100, 200, and 500 mg l
-1
) and expressed as
% inhibition.
50 mg l
-1
100 mg l
-1
200 mg l
-1
500 mg l
-1
Aglaothamnion pseudobyssoides 33.85
"
2.64 (c, d) 52.53
"
0.26 (d) 68.41
"
0.63 (c) 79.42
"
0.15 (b)
Asparagopsis armata 28.56
"
1.13 (e, f) 42.77
"
1.50 (e, f) 44.41
"
1.79 (g, h) 65.73
"
0.40 (h, i)
Brongniartella byssoides 74.86
"
2.34 (b) 86.21
"
0.11 (c) 91.65
"
0.17 (b) 94.80
"
0.08 (a)
Calliblepharis jubata 10.51
"
1.57 (h) 21.50
"
2.12 (l) 43.73
"
1.28 (h) 68.10
"
1.00 (f–h)
Callithamnion tetragonum 24.71
"
1.80 (f) 37.27
"
0.83 (f, g) 45.42
"
0.36 (f–h) 73.17
"
0.46 (c, d)
Callophyllis laciniata 19.26
"
1.26 (g) 31.72
"
0.52 (h, i) 47.63
"
0.31 (f, g) 68.93
"
0.39 (e–g)
Caulacanthus ustulatus 17.40
"
1.23 (g) 29.35
"
1.05 (i–k) 48.12
"
0.75 (f) 66.73
"
0.30 (g–i)
Ceramium ciliatum 18.75
"
2.04 (g) 30.95
"
1.95 (i, j) 46.08
"
2.37 (f–h) 66.15
"
1.75 (h, i)
Cystoclonium purpureum 15.74
"
1.53 (g) 26.06
"
0.68 (k) 42.79
"
0.71 (h) 62.23
"
0.18 (j, k)
Dilsea carnosa 27.00
"
0.64 (e, f) 34.82
"
0.51 (g, h) 51.81
"
0.48 (e) 70.68
"
0.30 (d–f)
Dumontia contorta 19.28
"
1.42 (g) 29.95
"
0.62 (i, j) 48.11
"
0.90 (f) 65.78
"
1.78 (h, i)
Furcellaria fastigiata 24.23
"
1.71 (f) 43.52
"
1.42 (e, f) 60.53
"
1.55 (d) 75.19
"
0.79 (c)
Gastroclonium ovatum 11.11
"
0.00 (h) 17.39
"
0.77 (m) 32.73
"
1.36 (j) 52.05
"
0.52 (l)
Gracilaria gracilis 18.32
"
1.36 (g) 28.29
"
0.60 (i–k) 44.69
"
1.89 (f–h) 62.78
"
0.58 (j, k)
Grateloupia filicina 2.40
"
1.01 (i) 14.27
"
1.36 (m) 32.74
"
2.10 (j) 51.47
"
1.28 (l)
Heterosiphonia plumosa 28.12
"
0.56 (e, f) 46.15
"
0.31 (e) 61.11
"
0.28 (d) 75.15
"
0.31 (c)
Kallymenia reniformis 1.69
"
0.00 (i) 9.33
"
2.42 (n) 20.18
"
0.64 (l) 42.75
"
0.86 (m)
Lomentaria articulate 9.98
"
1.80 (h) 17.68
"
0.87 (m) 35.09
"
0.54 (j) 50.89
"
1.10 (l)
Lomentaria clavellosa 16.08
"
1.45 (g) 27.72
"
0.62 (j, k) 38.65
"
0.44 (i) 64.21
"
0.46 (i, j)
Plocamium cartilagineum 30.07
"
1.92 (d, e) 40.09
"
0.48 (f, g) 52.20
"
0.31 (e) 70.85
"
0.41 (d, e)
Polyneura bonnemaisonii 8.72
"
1.07 (h) 10.52
"
1.03 (n) 23.56
"
1.95 (k) 38.97
"
2.05 (n)
Polysiphonia lanosa 38.16
"
1.17 (c) 55.65
"
0.40 (d) 66.21
"
0.60 (c) 80.88
"
0.72 (b)
Porphyra leucosticta 16.18
"
2.09 (g) 25.44
"
2.27 (k) 44.23
"
0.92 (g, h) 61.00
"
1.82 (k)
Porphyra purpurea 25.37
"
0.00 (f) 28.56
"
1.02 (i–k) 44.03
"
0.36 (h) 62.59
"
0.16 (j, k)
BHA 87.42
"
0.07 (a) 94.02
"
0.08 (a) 94.17
"
0.08 (a) 95.64
"
0.02 (a)
BHT 84.63
"
0.37 (a) 90.18
"
0.17 (b) 93.65
"
0.17 (a) 94.93
"
0.11 (a)
Ascorbic acid 90.04
"
0.32 (a) 92.91
"
0.14 (a, b) 93.53
"
0.09 (a) 94.87
"
0.01 (a)
a-Tocopherol 77.02
"
0.39 (b) 85.08
"
0.16 (c) 90.30
"
0.13 (b) 94.61
"
0.04 (a)
Values are means
"
SD (ns3). Significant differences determined by the Tukey HSD test (p-0.05) are indicated by different letters
(a–n).
Table 3 Antioxidant activities as % inhibition in red algal crude extracts at different concentrations (50, 100, 200, and 500 mg l
-1
)
assayed by the b-carotene-linoleic acid system.
50 mg l
-1
100 mg l
-1
200 mg l
-1
500 mg l
-1
Aglaothamnion pseudobyssoides 18.04
"
1.12 (d) 31.53
"
1.00 (f) 39.90
"
0.83 (e) 41.89
"
0.58 (j)
Asparagopsis armata 8.02
"
1.19 (h–j) 7.95
"
1.33 (l, m) 5.49
"
0.74 (p) 4.92
"
0.83 (o)
Brongniartella byssoides 75.90
"
0.74 (c) 83.86
"
1.42 (b, c) 89.33
"
1.03 (b) 91.40
"
0.51 (b)
Calliblepharis jubata 15.01
"
0.61 (d–f) 28.11
"
1.57 (f–h) 39.73
"
1.21 (e) 47.45
"
1.11 (f–h)
Callithamnion tetragonum 16.83
"
1.03 (d) 30.35
"
1.32 (f, g) 38.24
"
1.08 (e, f) 47.40
"
1.99 (f–h)
Callophyllis laciniata 7.84
"
0.70 (h–j) 16.00
"
0.49 (j) 22.48
"
0.88 (j, k) 28.48
"
0.80 (m)
Caulacanthus ustulatus 8.80
"
0.74 (h, i) 26.40
"
0.79 (g, h) 38.94
"
0.97 (e, f) 51.18
"
0.78 (e, f)
Ceramium ciliatum 12.18
"
0.78 (f, g) 25.82
"
1.76 (h) 36.02
"
0.41 (f, g) 46.22
"
1.44 (g–i)
Cystoclonium purpureum 8.15
"
1.01 (h–j) 16.00
"
0.58 (j) 18.49
"
0.56 (l, m) 20.45
"
0.77 (n)
Dilsea carnosa 9.43
"
0.27 (g–i) 20.98
"
1.51 (i) 31.77
"
1.19 (h, i) 42.99
"
0.37 (i, j)
Dumontia contorta 8.61
"
0.49 (h–j) 15.87
"
0.33 (j) 31.81
"
0.34 (h, i) 49.76
"
0.61 (f, g)
Furcellaria fastigiata 6.00
"
1.01 (j, k) 6.91
"
0.32 (l, m) 15.39
"
0.40 (n, o) 33.33
"
0.57 (k)
Gastroclonium ovatum 7.26
"
1.25 (i, j) 18.81
"
0.97 (i, j) 33.96
"
2.08 (g, h) 45.09
"
1.26 (h–j)
Gracilaria gracilis 4.37
"
0.12 (k) 7.31
"
0.14 (l, m) 18.31
"
0.45 (l–n) 33.16
"
1.41 (k, l)
Grateloupia filicina 7.08
"
0.02 (i, j) 12.36
"
1.15 (k) 24.44
"
0.30 (j) 33.96
"
2.69 (k)
Heterosiphonia plumosa 16.28
"
0.18 (d, e) 26.48
"
0.98 (g, h) 34.23
"
0.49 (g, h) 42.13
"
1.07 (i, j)
Kallymenia reniformis 1.93
"
0.88 (l) 5.61
"
0.77 (m) 15.91
"
0.59 (m–o) 20.60
"
0.83 (n)
Lomentaria articulata 10.33
"
1.80 (g, h) 17.34
"
2.68 (i, j) 20.25
"
1.80 (k, l) 27.26
"
1.58 (m)
Lomentaria clavellosa 12.66
"
1.15 (e–g) 20.13
"
1.26 (i) 24.80
"
1.31 (j) 34.05
"
2.82 (k)
Plocamium cartilagineum 5.97
"
0.63 (j, k) 16.26
"
0.25 (j) 22.08
"
0.90 (j, k) 29.05
"
0.67 (l, m)
Polyneura bonnemaisonii 6.13
"
0.42 (i–k) 9.44
"
0.45 (k, l) 14.58
"
0.18 (o) 20.70
"
1.71 (n)
Polysiphonia lanosa 24.08
"
0.90 (c) 47.92
"
1.34 (d) 61.16
"
0.49 (c) 66.75
"
0.72 (c)
Porphyra leucosticta 18.60
"
0.25 (d) 37.48
"
1.38 (e) 51.09
"
0.27 (d) 61.45
"
0.30 (d)
Porphyra purpurea 0.59
"
0.15 (m) 6.46
"
0.37 (m) 30.52
"
1.08 (i) 54.16
"
1.12 (e)
BHA 82.40
"
1.92 (b) 87.18
"
1.19 (a, b) 87.45
"
1.20 (b) 90.45
"
1.55 (b)
BHT 80.44
"
1.41 (b) 80.70
"
1.76 (c) 87.41
"
0.88 (b) 91.06
"
1.88 (b)
a-Tocopherol 89.01
"
1.39 (a) 90.16
"
0.18 (a) 93.42
"
0.59 (a) 97.20
"
0.54 (a)
Values are means
"
SD (ns3). Significant differences determined by the Tukey HSD test (p-0.05) are indicated by different letters
(a–p).
M. Zubia et al.: Biological activities of some red algae 273
Article in press - uncorrected proof
Table 4 Total phenolic content of red algal crude extracts
(means
"
SD; ns3) expressed in % of dry weight (% dw).
Species % dw
Aglaothamnion pseudobyssoides 5.73
"
0.04
Asparagopsis armata 1.13
"
0.05
Brongniartella byssoides 3.45
"
0.01
Calliblepharis jubata 1.74
"
0.03
Callithamnion tetragonum 1.85
"
0.03
Callophyllis laciniata 2.77
"
0.10
Caulacanthus ustulatus 2.77
"
0.09
Ceramium ciliatum 1.82
"
0.03
Cystoclonium purpureum 0.89
"
0.03
Dilsea carnosa 1.89
"
0.07
Dumontia contorta 0.56
"
0.01
Furcellaria fastigiata 3.25
"
0.02
Gastroclonium ovatum 0.73
"
0.03
Gracilaria gracilis 2.11
"
0.06
Grateloupia filicina 2.51
"
0.11
Heterosiphonia plumosa 3.29
"
0.03
Kallymenia reniformis 1.65
"
0.01
Lomentaria articulata 1.07
"
0.01
Lomentaria clavellosa 1.85
"
0.03
Plocamium cartilagineum 3.30
"
0.09
Polyneura bonnemaisonii 0.49
"
0.01
Polysiphonia lanosa 3.64
"
0.06
Porphyra leucosticta 2.05
"
0.01
Porphyra purpurea 1.32
"
0.06
plumosa (4.03
"
0.06 mg ml
-1
) provided the next most
active extracts (Figure 1).
The reducing activity assay confirmed the results
obtained by the DPPH assay. Table 2 shows that the
Brongniartella byssoides extract had the highest reducing
activity (94.80
"
0.08% at 500 mg l
-1
) among the species,
and its activity elicited a typical dose response. More-
over, statistical analysis showed that at 500 mg ml
-1
,
reducing activity of this extract was equivalent to the
reducing activities of any commercial antioxidants tested
and to that of a-tocopherol at lower concentrations. The
crude extracts from Polysiphonia lanosa, Aglaothamnion
pseudobyssoides, Furcellaria fastigiata, and Heterosipho-
nia plumosa also had high reducing activities (80.88
"
0.72%, 79.42
"
0.15%, 75.19
"
0.79%, and 75.15
"
0.31%
at 500 mg l
-1
, respectively), and this finding is in agree-
ment with the DPPH assay results. The correlation
between DPPH radical-scavenging activities and the
reducing activities measured in red algae extracts was
found to be significant (rs-0.843).
Table 3 shows that the strong antioxidant activity of
Brongniartella byssoides extract obtained in the previous
assays was confirmed by the b-carotene-linoleic acid
system assay, with this species having the highest per-
cent inhibition of b-carotene bleaching (91.40
"
0.51% at
500 mg l
-1
). The antioxidant activity of B. byssoides
extract was not significantly different from those of BHA
and BHT at concentrations of 500, 200, and 100 mg l
-1
.
In the case of Polysiphonia lanosa, our finding of a high
antioxidant activity (66.75
"
0.72% at 500 mg l
-1
) is con-
sistent with the results of the DPPH and reducing activity
assays. On the other hand, for Aglaothamnion pseudo-
byssoides, Furcellaria fastigiata, and Heterosiphonia plu-
mosa extracts, the results of the b-carotene-linoleic acid
system assay were not in full agreement with those from
the two other assays; the correlations between DPPH
radical-scavenging activities, or reducing activities, and
the antioxidant activities measured with the b-carotene-
linoleic acid system (rs-0.518 and rs0.596, respectively)
were rather low, although they were significant. However,
with this method, the antioxidant activities in the crude
extracts (500 mg l
-1
)fromPorphyra species were quite
high (61.45
"
0.30% and 54.16
"
1.12%).
Total phenolic content
Table 4 shows that the total phenolic contents of the
seaweed extracts varied from 0.49% dw (Polyneura
bonnemaisonii) to 5.73% dw (Aglaothamnion pseudobys-
soides). It is worth noting that the extracts with the high-
est antioxidant activities measured with the DPPH and
reducing activity assays, i.e., A. pseudobyssoides, Poly-
siphonia lanosa, Brongniartella byssoides, Heterosipho-
nia plumosa, and Furcellaria fastigiata, were also the
richest in phenolic compounds (5.73
"
0.04% dw,
3.64
"
0.06% dw, 3.45
"
0.01% dw, 3.29
"
0.03% dw,
3.25
"
0.02% dw, respectively). Indeed, significant corre-
lations were found between the total phenolic contents
and the DPPH radical-scavenging activities (rs-0.645)
and the reducing activities (rs0.665) measured in the
extracts. In contrast, there was no significant correlation
between phenolic contents and antioxidant activities
measured by the b-carotene assay (rs0.333).
Cytotoxic activity
The cytotoxic activities of the crude extracts were tested
against three cancer cell lines (Daudi, Jurkat, and K562).
Figure 2 illustrates the results of the tests and highlights
five crude extracts with significant cytotoxic activities
against Daudi cell lines (p-0.01): Asparagopsis armata
(32.52
"
7.33%), Brongniartella byssoides (57.92
"
12.65%), Heterosiphonia plumosa (60.89
"
10.89%), Plo-
camium cartilagineum (63.49
"
14.64%), and Ceramium
ciliatum (83.91
"
4.92%). Jurkat cell viability was signifi-
cantly decreased in treatments with extracts from A.
armata (30.07
"
7.24%, ps0.0000), B. byssoides
(38.31
"
8.35%, ps0.0000), H. plumosa (47.60
"
18.24%,
ps0.0009), and Calliblepharis jubata (76.74
"
12.74%,
ps0.0066). For K562 cell lines, only the B. byssoides
extract had significant cytotoxic activity (77.35
"
9.08%,
ps0.0016).
No significant correlation was found between phenolic
content and cytotoxic activities in the red algae under
study for all cancer cell lines (rs0.130, rs0.128, and
rs0.079 for Daudi, Jurkat, and K562 cell lines,
respectively).
Discussion
The antioxidative activities of red algal crude extracts
were characterized by three complementary biochemical
methods to overcome the inability of a simple one-
dimensional test of antioxidant capacity to accurately
mirror the complex in vivo interactions between antioxi-
dants (Frankel and Meyer 2000, Huang et al. 2005).
DPPH, reducing activity, and b-carotene-linoleic acid
system assays are known to be simple, fast, and reliable,
and they proved their relevance here in assessment of
274 M. Zubia et al.: Biological activities of some red algae
Article in press - uncorrected proof
Figure 2 Percentage cell viability following treatment and in a control (means
"
SD, ns6).
Three different cancer cell lines (Daudi, Jurkat, and K562) were exposed for 24 h to the red algal crude extracts (a key to the red
algal extract numbers is given in Table 1) and to the control (DMSO, 100
mgml
-1
). * Indicates significant differences (p-0.01) compared
to the control.
the total antioxidant activity of the extracts. The finding
of a significant correlation between the DPPH radical-
scavenging activities and the reducing activities dis-
played by the red algal extracts under study is explained
by the fact that both assays rely on electron/hydrogen
donation. On the other hand, the emulsified lipid used in
the b-carotene assay introduced additional variables that
affect the oxidation process in comparison to the other
methods. In the more complex b-carotene system, anti-
oxidant molecules may inhibit lipid oxidation by several
mechanisms (e.g., prevention of chain initiation, binding
of transition metal ion catalysts, decomposition of per-
oxides) in addition to free radical trapping (Frankel and
Meyer 2000).
Among the species under study, Brongniartella bys-
soides had the highest antioxidant activity in all assays,
and its antioxidant activity was equivalent to commercial
antioxidant compounds tested in this experiment: ascor-
bic acid, a-tocopherol, BHA and BHT. To the best of our
knowledge, this study is the first report to provide evi-
dence of high antioxidant activity in B. byssoides.
Although this species belongs to the order Ceramiales,
M. Zubia et al.: Biological activities of some red algae 275
Article in press - uncorrected proof
which is the subject of extensive studies due to its huge
variety of bioactive compounds (mainly halogenated,
e.g., terpenoids, bromophenols, acetogenins; Korn-
probst 2005), literature data are scarce. In this study,
extracts of four species in the order Ceramiales, i.e.,
Aglaothamnion pseudobyssoides, B. byssoides, Hetero-
siphonia plumosa, and Polysiphonia lanosa, were among
the five most active. This finding constitutes additional
evidence for the bioactive potential of this order. More-
over, it is worth noting that both B. byssoides and P. lanosa
belong to the family Rhodomelaceae. Numerous chemi-
cal investigations have led to the isolation of an impres-
sive array of bioactive compounds in this family,
especially halogenated terpenoids in the genus Laurencia
(Kornprobst 2005). Few studies have evidenced a
marked antioxidant activity in Polysiphonia extracts (Fuji-
moto 1990, Yan et al. 1998, Dummermuth et al. 2003),
and some bromophenols isolated from Polysiphonia
urceolata have been identified as the antioxidant mole-
cules (Fujimoto 1990, Duan et al. 2006, Li et al. 2007,
2008). Bromophenols from P. lanosa have also demon-
strated antimicrobial (Glombitza et al. 1985, Fariq 1991)
and cytotoxic (Shoeib et al. 2004) activities. These
results, as well as the finding here of a high phenolic
content in B. byssoides and P. lanosa extracts, suggest
that bromophenols might be responsible for the antioxi-
dant activity of these extracts.
Aglaothamnion pseudobyssoides and Heterosiphonia
plumosa also proved to be active, but there is a lack of
published information about their antioxidant capacities.
Our study suggests that the antioxidant mechanisms at
play in these extracts rely mainly on the trapping of a free
radical, because the strong antioxidant activity exhibited
by these extracts was not confirmed by the b-carotene
assay. This result might also be explained by the inter-
actions (synergistic, additive or antagonistic effects)
between the emulsified medium used in the b-carotene
assay and the complex composition of the crude
extracts. The high phenolic contents measured in A.
pseudobyssoides (5.73% dw) and H. plumosa (3.29%
dw) extracts could partly explain their high radical-scav-
enging activity. This finding is in agreement with a pre-
vious study (Zubia et al. 2007), which showed that
marked DPPH radical-scavenging activity correlated with
high phenolic content in a Heterosiphonia extract. In fact,
phenolic compounds are thought to protect the algal
thallus against photodestruction by UV radiation and to
have radical-scavenging properties (Connan et al. 2006).
Numerous studies have demonstrated a highly significant
correlation between phenolic content and antioxidant
activity in seaweed extracts (e.g., Kim et al. 2005, Zhang
et al. 2007), as confirmed in this study (rs-0.645 and
0.665). However, in this study, the highest antioxidant
activity found in Brongniartella byssoides extract did not
correspond to the highest phenolic content (found in A.
pseudobyssoides extract). A possible explanation may be
that there are other types of antioxidant molecules struc-
turally different from phenolic compounds in red algae
(e.g., carotenoids, terpenoids, ascorbic acid).
This study also highlighted marked antioxidant activity
in Furcellaria fastigiata, which belongs to the order Gigar-
tinales. The high phenolic content in the F. fastigiata
extract (3.25% dw) suggests that phenolic compounds
may be responsible for the antioxidant activity. Indeed,
some bioactive halogenated phloroglucinols and nitro-
genous derivative compounds have been previously
identified in carrageenophyte species, such as Furcellaria
(Kornprobst 2005). There is a lack of available literature
on its bioactivity, but our preliminary result would encour-
age additional investigations on this species because it
is easily cultivated. Sufficient amounts of cultured algae
with similar biochemical and chemical characteristics
could be provided for purification and isolation of the
bioactive molecules, and the traceability could be
ensured.
With the b-carotene system assay, Porphyra leucostic-
ta and Porphyra purpurea extracts showed some specific
antioxidant activity. As Porphyra, commonly known as
nori or laver, is widely used in Asia as part of the human
diet, it has been the subject of numerous studies per-
formed in order to identify its functional properties. Sev-
eral antioxidant molecules have been identified in the
genus Porphyra: histidine-related compounds (Tamura et
al. 1998), chlorophyll analogs and mycosporine-like ami-
no acids (Nakayama et al. 1999), sulfated polysaccha-
rides (Zhang et al. 2003, 2004) and oligosaccharides (Wu
and Pan 2004). It has been suggested that the accu-
mulation of the UV-absorbing mycosporine-like amino
acids, such as porphyra-344, provides a photoprotection
to P. leucosticta, and thus these compounds may func-
tion as biological antioxidants (Dunlap and Yamamoto
1995, Korbee et al. 2005).
This study highlighted the strong cytotoxicity of crude
extracts from Asparagopsis armata, Brongniartella bys-
soides, and Heterosiphonia plumosa against the human
cancer cell lines Daudi and Jurkat. This is the first report
of cytotoxic activities for these red macroalgae. The
literature contains reports of antimicrobial activity of
halogenated metabolites produced by A. armata (Korn-
probst 2005), but until now there has been no evidence
of its cytotoxic activity. This screening has also confirmed
the great bioactivity of species in the Ceramiales, espe-
cially B. byssoides, which had cytotoxic activities against
all human cancer cell lines tested. Numerous cytotoxic
compounds have been isolated previously from species
of Ceramiales, especially from the genus Laurencia (e.g.,
Pec et al. 2003, Mohammed et al. 2004, Sun et al. 2005),
and a promising anticancer drug (dehydrothyrsiferol)
extracted from Laurencia viridis has been developed to
the preclinical phase (Pec et al. 2003). Phenolic com-
pounds, such as bromophenols, have also been identi-
fied as the cytotoxic molecules in Rhodomela confer-
voides (Hudson) P.C. Silva (Xu et al. 2004), but no signif-
icant correlation was found in this study between cyto-
toxic activities and phenolic content. The ability of
phenols to protect cells from oxidative stress has been
demonstrated, but these compounds have a contradic-
tory behavior characterized by anti- and protumoral
activities according to their chemical structure, the sys-
tem and conditions used in the study (Gomes et al.
2003). Further purifications are required to isolate and
identify the cytotoxic molecules in A. armata, B. bys-
soides, and H. plumosa extracts.
The finding of a positive correlation between cytotoxic
effects and the strong DPPH radical-scavenging activity
276 M. Zubia et al.: Biological activities of some red algae
Article in press - uncorrected proof
in Brongniartella byssoides extract makes this species a
promising candidate for further investigations. Oxidative
stress is known to be implicated in the process of car-
cinogenesis through damage to cellular molecules, such
as proteins, lipids and nucleic acids. Hence, the preva-
lence of both antioxidant and cytotoxic properties in a
single compound could be beneficial in terms of rational,
preventive or therapeutic purposes. However, additional
studies are needed to demonstrate that this extract
exhibits no cytotoxicity towards normal cells. Detailed
examination of the mechanism of action of the isolated
and purified compounds of B. byssoides extract should
be investigated, e.g., their impact on cell cycle, their abil-
ity to activate caspases or induce mitochondrial and DNA
damages.
Conclusion
This study constitutes the largest screening of antioxi-
dant and cytotoxic activities in red macroalgae from the
Brittany coasts to date. The results clearly indicate that
extracts of the 24 species of red algae tested possess
antioxidant activity to varying degrees. This screening
emphasized the great antioxidant potential of Brongniar-
tella byssoides, which was found to be equivalent to the
antioxidant activity of commercial antioxidant molecules
assessed in the same study. Moreover, three other Cera-
miales species (Aglaothamnion pseudobyssoides, Hete-
rosiphonia plumosa, and Polysiphonia lanosa) had high
antioxidant activities, as did one member of the Gigarti-
nales (Furcellaria fastigiata) and two species of Bangiales
(Porphyra leucosticta and Porphyra purpurea). The cor-
relation usually found between marked radical-scaveng-
ing activities and a high total phenolic content supports
the involvement of phenolic compounds in the antioxi-
dant mechanisms. Furthermore, extracts from Aspara-
gopsis armata, B. byssoides and H. plumosa showed a
strong cytotoxicity against human cancer cell lines. Sup-
plementary to these findings, it would be worth while car-
rying out additional experiments on the identification and
characterization of the bioactive compounds of the most
active extracts, especially from B. byssoides, and on the
understanding of their mechanisms of action.
Acknowledgements
The authors thank Erwan Ar-Gall and Michel le Duff (LEBHAM/
EA3877/IUEM/UBO) for their help in identifying the macroalgal
species and Marie-Paule Friocourt for her help in correcting the
English of this paper.
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Received 12 September, 2008; accepted 4 February, 2009;
online first 27 April, 2009
