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Antioxidant activities in tropical marine macroalgae
from the Yucatan Peninsula, Mexico
Mayalen Zubia &Daniel Robledo &
Yolanda Freile-Pelegrin
Received: 19 July 2006 /Accepted: 16 November 2006 / Published online: 27 February 2007
#Springer Science + Business Media B.V. 2007
Abstract Extracts from 48 marine macroalgae species (17
Chlorophyta, 8 Phaeophyta and 23 Rhodophyta) from the
coasts of Yucatan and Quintana Roo (Mexico) were
evaluated for antioxidant activity. The antioxidant activity
was measured with the DPPH (2,2-diphenyl-1-picrylhydra-
syl) method, and the phenolic content of each extract were
also evaluated. All species exhibited a DPPH radical
scavenging activity, and three species (Avrainvillea long-
icaulis,Chondria baileyana and Lobophora variegata)
demonstrated great antioxidant potential with very low
oxidation index EC
50
(1.44±0.01, 2.84±0.07 and 0.32 ±
0.01 mg mL
−1
, respectively), significantly equivalent to
EC
50
of some commercial antioxidants such as α-tocoph-
erol, ascorbic acid, BHA and BHT. Moreover, extracts of
themostactivespeciesexhibited reducing activities,
superoxide anion radical scavenging and inhibition of lipid
peroxidation. These results suggest that some macroalgae
from the Yucatan peninsula have a great antioxidant
potential which could be considered for future applications
in medicine, food production or cosmetic industry.
Key words antioxidant activity .macroalgae
Introduction
In the past decade, the search for natural antioxidant
compounds has gained considerable attention and the
number of publications on antioxidants and oxidative stress
has nearly quadrupled (Huang et al. 2005). Antioxidant
compounds play an important role against various diseases
(e.g., chronic inflammation, atherosclerosis, cancer and
cardiovascular disorders) and ageing processes (Kohen and
Nyska 2002), which explains their considerable commercial
potential in medicine, food production and the cosmetic
industry. Moreover, interest in employing antioxidants from
natural sources is considerably enhanced by consumer
preference for natural products and concern about the
potential toxic effects of synthetic antioxidants (Safer and
al-Nughamish 1999).
Marine algae, like other photosynthesizing plants, are
exposed to a combination of light and oxygen that leads to
the formation of free radicals and other strong oxidizing
agents. However, the absence of oxidative damage in the
structural components of macroalgae (i.e., polyunsaturated
fatty acids) and their stability to oxidation during storage
suggest that their cells have protective antioxidative defense
systems (Fujimoto 1990; Matsukawa et al. 1997). In fact,
algae have protective enzymes (superoxide dismutase,
peroxidase, glutathione reductase, catalase) and antioxida-
tive molecules (phlorotannins, ascorbic acid, tocopherols,
carotenoids, phospholipids, chlorophyll related compounds,
bromophenols, catechins, mycosporine-like amino acids,
polysaccharides, etc.) which are similar to those of vascular
plants (Fujimoto 1990; Le Tutour et al. 1998; Rupérez et al.
2002; Yuan et al. 2005).
Antioxidative properties of seaweed extracts have been
studied in several geographic regions, but only a few
studies have been performed on tropical seaweed species
(Anggadiredja et al. 1997; Lim et al. 2002; Fallarero et al.
2003; Santoso et al. 2004). Lack of information about the
antioxidant activity of tropical macroalgae is surprising
since these species are expected to develop a very effective
antioxidant defence system due to the strong UV radiation
in the tropical environment. In fact, previous studies have
J Appl Phycol (2007) 19:449–458
DOI 10.1007/s10811-006-9152-5
M. Zubia :D. Robledo :Y. Freile-Pelegrin (*)
Departamento de Recursos del Mar, CINVESTAV Unidad Mérida,
Km 6 Carretera Antigua a Progreso, A.P. 73 Cordemex,
97310 Mérida, Yucatan, México
e-mail: freile@mda.cinvestav.mx
demonstrated that UV radiation induces the promotion of
antioxidant defense in macroalgae (Aguilera et al. 2002;
Bischof et al. 2002).IntheGulfofMexicoandCaribbean
coast of Yucatan and Quintana Roo, there are numerous species
ofmacroalgaeexposedtohighsolar irradiation and some of
them have never been studied for their antioxidant activities.
The present study evaluated the antioxidative potential
of 48 species of macroalgae from the coasts of Yucatan and
Quintana Roo (Mexico) by measuring the 2,2-diphenyl-1-
picrylhydrasyl (DPPH) radical scavenging activity and total
content of phenolic compounds in alcoholic extracts.
Antioxidant activities have been attributed to various
reactions and mechanisms: prevention of chain initiation,
binding of transition metal ion catalysts, reductive capacity,
radical scavenging, etc. (Frankel and Meyer 2000; Huang
et al. 2005). Therefore, in order to better understand the
antioxidant processes involved, the specific antioxidative
activities of the most active extracts were also character-
ized, using different biochemical methods: reducing activ-
ity, superoxide anion scavenging activity and inhibition of
lipid peroxidation.
Materials and methods
Collection and preparation of algal extracts
Forty-eight species of macroalgae were collected from the
Gulf of Mexico and Caribbean coast of Yucatan and
Quintana Roo (Fig. 1) between October 2005 and February
2006. Once harvested, macroalgae were stored in plastic
bags and placed on ice for transport to the laboratory.
Voucher specimens of all species were pressed and stored in
4% formol for identification according to Wynne (2005).
Samples were washed thoroughly with fresh water to
remove salts, sand and epiphytes, and stored at −20°C.
Entire plants of each macroalgae were lyophilized and
milled into powder before extraction. Lyophilized samples
(5 g) were extracted twice with 75 mL of dichloromethanol:
methanol (2:1) during 20 h. Extracts were combined,
filtered and concentrated under reduced pressure to 10 mL,
and stored at −20°C.
Determination of phenolic content
Total content of phenolic compounds of algal extracts was
determined spectrophotometrically using Folin-Ciocalteu
reagent according to the method described in Lim et al.
(2002). First, extracts (0.3 mL) were diluted with methanol
(2.7 mL). Aliquots of the diluted extracts (0.1 mL) were
transferred into the test tubes; 2.9 mL of distilled water
and 0.5 mL of Folin-Ciocalteu reagent were added. After
10 min, 1.5 mL of 20% sodium carbonate solution was
added and the mixture was mixed thoroughly and allowed
to stand at room temperature in the dark for 1 h. Ab-
sorbance was measured at 725 nm and total content of
phenolic compounds was calculated based on a stan-
dard curve of phloroglucinol and expressed in % of dry
weight.
DPPH radical scavenging activity
DPPH (2,2-diphenyl-1-picrylhydrasyl) radical scavenging
activity was determined according to the method of Brand-
Williams et al. (1995). Briefly, 100 μL of each extract at
various dilutions (pure, 0.5, 0.25, etc., depending on the
activity of each extract) were mixed with 3.9 mL of a DPPH
solution (25 mg L
−1
) prepared daily. Due to color intensity
of the extracts, it was necessary to prepare a blank of 100 μL
of each extract at various dilutions added to 3.9 mL of
methanol. The reaction was complete after 2 h in the dark at
room temperature, and the absorbance was read at 515 nm.
DPPH versus absorbance was calculated by linear regres-
sion (n=8; r=0.99): [DPPH]=36.76 (Abs+0.0044). These
values were plotted against percentage of DPPH (% DPPH)
to estimate 50% reduction of its initial value (EC
50
;
efficient concentration, also called oxidation index). Every
sample was done in triplicate and mean values were
obtained to calculated the EC
50
. Positive controls such as
ascorbic acid, α-tocopherol, BHT (butylated hydroxyto-
luene) and BHA (butylated hydroxyanisole) were also
measured, and the means of the EC
50
of each control were
calculated from five measurements.
Fig. 1 Map of Yucatan peninsula indicating the collecting sites in
Yucatan (1Telchac; 2Dzilam de Bravo) and Quintana Roo (3Cancún;
4Playa del Carmen; 5Tulum) coasts
450 J Appl Phycol (2007) 19:449–458
Reducing activity
Reducing activity of the extracts was evaluated according
to the method of Oyaizu (1986) in Yen and Chen (1995).
This assay measures the total antioxidant capacity of an
extract evaluating the redox potentials of the compounds.
Extract samples (0.5 mL) at two different concentrations
(0.1 and 0.5 mg mL
−1
) were mixed with phosphate buffer
(1.25 mL, 0.2 M, pH 6.6) and potassium ferricyanide (K
3
Fe
(CN)
6
; 1.25 mL, 1%) and incubated at 50°C for 20 min,
cooled and mixed with 1.25 mL of trichloroacetic acid
(10%), and 1.25 mL of this mixture was transferred to other
test tubes in which distilled water (1.25 mL) and
FeCl
3
.6H
2
O (0.25 mL, 0.1%) were added. The mixture
was centrifuged and kept at room temperature for 10 min
before reading the absorbance at 700 nm. Every sample and
positive controls (ascorbic acid, α-tocopherol, BHT and
BHA) were done in triplicate.
Superoxide anion radical scavenging activity
Superoxide anion is one of the most effective free radical,
implicated in cell damage as precursors of mainly reactive
oxygen species, contributing to the pathological process of
many diseases. The superoxide anion scavenging activity of
the extracts was determined using a non enzymatic system
described in Lim et al. (2002). All reagents were prepared
with Tris-HCl buffer (16 mM, pH 8.0). In the test tubes,
1 mL of 234 μM NADH, 1 mL of 150 μM nitroblue
tetrazolium (NBT) and 0.2 mL of the seaweed extracts at
three concentrations (0.1, 0.5 and 1 mg mL
−1
) were mixed
together with 0.8 mL of 37.5 μM phenazine methosulfate
(PMS); after 5 min the absorbance was measured at
560 nm. The same mixture without sample extract was
used as control, and blanks for each extract were prepared
without PMS. Every sample and positive controls (acid
ascorbic, α-tocopherol and BHT) were done in triplicate.
Inhibition of lipid oxidation
Oxidation of unsaturated fatty acids in biological membranes
leads to the formation of lipid radicals and destruction of
lipid membranes. Antioxidants can disrupt the free-radical
chain reaction by donating their hydrogen to fatty acids
radicals to terminate chain reaction. The ferric thiocyanate
(FTC) method of Larrauri et al. (1996) in Sanchez-Moreno
et al. (1999) used a linoleic acid model to evaluate inhibition
of lipid oxidation. Sample extracts (0.5 mL, 0.5 mg mL
−1
)
were mixed with an emulsion of 2.51% linoleic acid in
absolute ethanol (0.5 mL), 0.05 M phosphate buffer pH 7
(1 mL) and distilled water (0.5 mL) in a screw capped tube,
shaken and incubated in an oven at 40°C in the dark. The
same mixture without sample extract was used as control. At
different time intervals (0–10 days), 0.1 mL of each tube was
transferred to the other tube where 9.7 mL of 75% ethanol
and 0.1 mL of 30% ammonium thiocyanate together with
0.1 mL of 0.02 M ferrous chloride in 3.5% hydrochloric acid
were added; after 3 min, the absorbance was measured at
500 nm. Every sample and positive controls (α-tocopherol,
BHT and BHA) were done in triplicate.
Statistical analysis
All statistical analyses were performed with Statistica 6
software. Data were tested for normality (Shapiro–Wilk’s
test) and subjected to Bartlett’s test to verify the homoge-
neity of variances groups. One-way analysis of variance
(ANOVA) was used to compare antioxidant activity
between extracts after transformation of data if necessary
and post-hoc test (Tukey HSD) was performed when data
showed significant differences (p<0.05).
Results and discussion
During the study, 48 species of macroalgae were collected
from the coasts of Yucatan and Quintana Roo: 17
Chlorophyta, 8 Phaeophyta and 23 Rhodophyta. The
species and their total phenolic content (% dry wt) are
listed in Table 1. DPPH radical scavenging activities,
expressed as oxidation index EC
50
(mg mL
−1
) for Chlor-
ophyta, Phaeophyta and Rhodophyta are summarized in
Figs. 2,3and 4, respectively.
Chlorophyta
All species of Chlorophyta collected showed antioxidant
activities (Fig. 2). Avrainvillea longicaulis exhibited the
highest activity with a very low EC
50
(1.44±0.01 mg
mL
−1
) significantly equivalent to EC
50
of two commercial
antioxidants tested, α-tocopherol (0.31 ± 0.03 mg mL
−1
)
and BHT (0.16±0.01 mg mL
−1
), in agreement with the
study of Takamatsu et al. (2003) who found a strong
antioxidant activity for this genus and isolated the
avrainvilleol, a brominated diphenylmethane derivative,
as the antioxidant compound. This molecule also exhibited
various biological activities as feeding-deterrent, antibac-
terial and ichthyocidal (Sun et al. 1983; Hay et al. 1990).
Although no isolation and characterization of the extracts
of A. longicaulis were done, the phenolic content in the
extract (3.36± 0.05% dry wt) (Table 1) may suggest the
presence of avrainvilleol.
Extracts of Halimeda tuna,Caulerpa cupressoides and
Caulerpa paspaloides exhibited also relatively high DPPH
radical scavenging activities (6.17± 0.10, 6.35 ±0.15 and
7.36±0.16 mg mL
−1
, respectively). Previous studies have
J Appl Phycol (2007) 19:449–458 451
Table 1 Phenolic content of the species collected in Quintana Roo
1
and the Yucatan
2
coasts of Mexico
Species Locality Phenolic content (% dry wt)
Chlorophyta
Acetabularia schenckii K. Möbius Cancun
1
0.76± 0.05
Avrainvillea longicaulis (Kützing) G. Murray & Boodle Puerto Morelos
1
3.36± 0.05
Caulerpa ashmeadii Harvey Dzilam de Bravo
2
1.42± 0.11
Caulerpa cupressoides (H. West in Vahl) C. Agardh Dzilam de Bravo
2
4.36± 0.31
Caulerpa paspaloides (Bory de Saint-Vincent) Greville Dzilam de Bravo
2
4.26± 0.21
Caulerpa prolifera (Forsskål) J.V. Lamouroux Dzilam de Bravo
2
8.13± 0.52
Caulerpa sertularioides (S.G. Gmelin) M. Howe Tulum
1
6.48± 0.17
Caulerpa taxifolia (H. West in Vahl) C. Agardh Tulum
1
6.60± 0.23
Cladophora prolifera (Roth) Kützing Playa de Carmen
1
1.95± 0.12
Cladophora vagabunda (Linnaeus) Hoek Cancun
1
1.02± 0.08
Codium decorticatum (Woodward) M. Howe Dzilam de Bravo
2
0.54± 0.04
Enteromorpha intestinalis (Linnaeus) Nees Cancun
1
1.42± 0.11
Halimeda monile (J. Ellis & Solander) J.V. Lamouroux Telchac
2
0.47± 0.00
Halimeda tuna (J. Ellis & Solander) J.V. Lamouroux Telchac
2
1.70± 0.02
Penicillus dumetosus (J.V. Lamouroux) Blainville Telchac
2
2.24± 0.06
Penicillus pyriformis A. Gepp & E.S. Gepp Telchac
2
1.35± 0.16
Udotea conglutinata (J. Ellis & Solander) J.V. Lamouroux Dzilam de Bravo
2
2.04± 0.21
Phaeophyta
Dictyota cervicornis Kützing Dzilam de Bravo
2
5.55± 0.23
Dictyota ciliolata Sonder ex Kützing Tulum
1
5.53± 0.09
Dictyota crenulata J. Agardh Dzilam de Bravo
2
1.99± 0.06
Lobophora variegata (J.V. Lamouroux) Womersley ex E.C. Oliveira Telchac
2
29.18± 0.32
Padina gymnospora (Kützing) Sonder Playa de Carmen
1
5.58± 0.30
Sargassum pteropleuron Grunow Telchac
2
0.76± 0.04
Sargassum ramifolium Kützing Telchac
2
0.95± 0.10
Turbinaria tricostata E.S. Barton Tulum
1
1.05± 0.08
Rhodophyta
Acanthophora spicifera (M. Vahl) Børgesen Telchac
2
1.19± 0.09
Bryothamnion triquetrum (S.G. Gmelin) M. Howe Tulum
1
1.55± 0.21
Ceramium nitens (C. Agardh) J. Agardh Tulum
1
1.92± 0.07
Champia salicornioides Harvey Cancun
1
2.64± 0.33
Chondria atropurpurea Harvey Tulum
1
3.21± 0.40
Chondria baileyana (Montagne) Harvey Cancun
1
7.30± 0.29
Chondrophycus papillosus (C. Agardh) Garbary & J.T. Harper Tulum
1
1.34± 0.17
Chondrophycus poiteaui (J.V. Lamouroux) K.W. Nam Puerto Morelos
1
1.77± 0.10
Digenea simplex (Wulfen) C. Agardh Telchac
2
0.64± 0.08
Eucheuma isiforme (C. Agardh) J. Agardh Telchac
2
0.36± 0.08
Gracilaria bursa-pastoris (S.G. Gmelin) P.C. Silva Telchac
2
0.38± 0.01
Gracilaria caudata J. Agardh Telchac
2
0.34± 0.03
Gracilaria cornea J. Agardh Telchac
2
0.50± 0.12
Gracilaria cylindrica Børgesen Telchac
2
0.41± 0.03
Gracilaria tikvahiae McLachlan Telchac
2
0.36± 0.02
Gracilariopsis tenuifrons (C.J. Bird & E.C. Oliveira) Fredericq & Hommersand Playa de Carmen
1
2.04± 0.13
Halymenia floresii (Clemente & Rubio) C. Agardh Telchac
2
1.03± 0.08
Heterosiphonia gibbesii (Harvey) Falkenberg Playa de Carmen
1
3.45± 0.36
Hypnea spinella (C. Agardh) Kützing Playa de Carmen
1
0.67± 0.06
Laurencia intricata J.V. Lamouroux Dzilam de Bravo
2
2.51± 0.10
Laurencia obtusa (Hudson) J.V. Lamouroux Tulum
1
4.17± 0.12
Liagora ceranoides J.V. Lamouroux Tulum
1
0.90± 0.10
Nemalion helminthoides (Velley) Batters Playa de Carmen
1
1.87± 0.03
452 J Appl Phycol (2007) 19:449–458
reported high antioxidant activity in the genus Halimeda
(Fallarero et al. 2003) and Caulerpa (Santoso et al. 2004;
Cavas and Yurdakoc 2005). High activity among the
Udoteaceae is not surprising as numerous chemical inves-
tigations have led to the isolation of an impressive array of
bioactive compounds, mainly sesquiterpenoid and diterpe-
noid metabolites (Fenical and Paul 1984). High phenolic
content has been measured in our study for Caulerpa
species, with C. prolifera showing a maximum of 8.13±
0.52% dry wt (Table 1). However, an overestimation may
likely occur with the Folin-Ciocalteu (FC) method because
of reaction with all oxidizing compounds with an aromatic
cycle (phenylalanine, tyrosine, tryptophane, ascorbic acid)
(Ragan and Glombitza 1986).
Lowest antioxidant activities were observed in Enter-
omorpha intestinalis and Codium decorticatum (43.23 ±
0.28 and 51.48±0.25 mg mL
−1
, respectively) in accordance
with previous published data on E. intestinalis (Yan et al.
0.06 (a)
0.09 (a)
0.16 (ab)
0.31 (ab)
1.44 (b)
6.17 (c)
6.35 (c)
7.36 (cd)
8.46 (de)
9.51 (ef)
9.52 (ef)
9.66 (ef)
10.31 (f)
10.64 (f)
14.39 (g)
15.32 (g)
16.66 (h)
19.28 (i)
23.16 (j)
43.23 (k)
51.48 (l)
0 10203040506
0
Codium decorticatum
Enteromorpha intestinalis
Cladophora vagabunda
Penicillus pyriformis
Cladophora prolifera
Caulerpa taxifolia
Caulerpa ashmeadii
Penicillus dumetosus
Halimeda monile
Caulerpa sertularioides
Acetabularia schenckii
Udotea conglutinata
Caulerpa prolifera
Caulerpa paspaloides
Caulerpa cupressoides
Halim eda tuna
Avrainvillea longicaulis
-tocopherol
BHT
Ascorbic acid
BHA
EC
50
(mg mL
-1
)
Fig. 2 DPPH radical scaveng-
ing activity expressed in oxida-
tion index EC
50
given in mg
mL
−1
(mean ± SD; n=3) for
Chlorophyta extracts and con-
trols (ascorbic acid, BHA, BHT
and α-tocopherol). Bars repre-
sent standard deviations. Signif-
icant differences are indicated
by different letters as deter-
mined by Tukey HSD test
(p<0.05)
0.32 (a)
0.31 (a)
0.16 (a)
0.09 (a)
3.45 (b)
0.06 (a)
6.42 (c )
6.64 (cd)
7.14 (d)
8.85 (e)
12.40 (f)
34.88 (g
)
0 1020304
0
Dictyota crenulata
Dictyota ciliolata
Turbinaria tricostata
Sargass um pt eropleuron
Sargassum ramif olium
Dict yota cervicornis
Padina gymnos pora
Lobophora variegata
-toc opherol
BHT
Ascorbic acid
BHA
EC
50
(m
g
mL
-1
)
Fig. 3 DPPH radical scaveng-
ing activity expressed in oxida-
tion index EC
50
given in mg
mL
−1
(mean ± SD; n=3) for
Phaeophyta extracts and con-
trols (ascorbic acid, BHA, BHT
and α-tocopherol). Bars repre-
sent standard deviations. Signif-
icant differences are indicated
by different letters as deter-
mined by Tukey HSD test
(p<0.05)
J Appl Phycol (2007) 19:449–458 453
1998; Kim et al. 2005) and in Codium fragile (Kim et al.
2005). This result is surprising because E. intestinalis lives
in highly dynamic upper littoral areas characterized by
numerous stress factors (i.e. high irradiance, temperature
and desiccation). Choo et al. (2004) defined E. ahlneriana
as a stress-susceptible species, and explained the lack of
antioxidant defence in its life strategy. Indeed, this species
is ephemeral and seems to prefer to allocate its resource to
fight against epiphytes rather than oxidative stress.
Phaeophyta
All species of Phaeophyta showed antioxidant activities
(Fig. 3). Lobophora variegata exhibited high DPPH as
shown by the lowest EC
50
(0.32±0.01 mg mL
−1
) signifi-
cantly comparable to EC
50
of all commercial antioxidants
tested: α-tocopherol (0.31±0.03 mg mL
−1
), BHT (0.16±
0.01 mg mL
−1
), ascorbic acid (0.09±0.02 mg mL
−1
) and
BHA (0.06±0.02 mg mL
−1
). There is no information avail-
able on the antioxidant activity of L. variegata, although
Kubanek et al. (2003) reported that crude extracts of L.
variegata have particularly strong antifungal bioactivity
and identified the cyclic lactone lobophorolide as a defen-
sive compound in preliminary experiments surveying 55
species of Caribbean macroalgae for antimicrobial poten-
tial. Moreover, it had the highest level of phenolic content
as tested in this study, with 29.18% dry wt (Table 1). In
general, phenolic compounds range between 20–30% dry
wt in brown algae (Ragan and Glombitza 1986). Targett
et al. (1995) reported a phenolic content of 25% dry wt in
L. variegata. These results suggest that the antioxidant
activity of L. variegata extracts could be correlated with
its high phenolic content. In fact, phenolic compounds are
considered to protect the algal thallus from photodestruc-
tion caused by UV radiation (Pavia et al. 1997)andto
exhibit radical scavenging properties (Rice-Evans et al.
1997). Several studies have demonstrated a highly signif-
icant correlation between the phenolic content and the
antioxidant activity in seaweed extracts (Siriwardhana et
al. 2003;Karawitaetal.2005; Kim et al. 2005;Yuanetal.
2005;Connanetal.2006). In addition, some studies have
described the antioxidant activity of some purified phenolic
compounds in Eisenia bicyclis (Nakamura et al. 1996)andin
Sargassum kjellmanianum (Yan et al. 1996; Wei et al. 2003).
0.31 (a)
0.16 (a)
0.09 (a)
0.06 (a)
2.84 (a)
8.15 (b)
8.86 (b)
10.43 (bc)
12.50 (cd)
12.65 (cde)
12.89 (cde)
13.49 (cde)
13.89 (de)
14.52 (de)
15.75 (ef)
17.84 (f)
21.36 (g)
27.34 (h)
28.94 (h)
33.73 (i)
37.50 (j)
42.27 (k)
43.52 (k)
52.56 (l)
62.82 (m)
72.51 (n)
77.71 (o
)
0 102030405060708090
Halymenia f loresii
Gracilaria c ornea
Gracilaria cylindrica
Laurencia i ntri cat a
Eucheuma isiforme
Gracil aria b ursa-pastori s
Laurencia obt usa
Gracil aria c audata
Gracilaria tikvahiae
Champia sal ic ornioides
Digenea simplex
Chondrophycus papillos us
Chondrophycus poi teaui
Hypnea spinell a
Ceramium nit ens
Nemalion helminthoides
Bryothamni on tri quetrum
Liagora ceranoi des
Acant hophora spi ci fera
Chondria atropurpurea
Gracil ariops is tenui frons
Heterosi phonia gi bb esi i
Chondria bai leyana
-tocopherol
BHT
Ascorbic acid
BHA
EC
50
(m
g
mL
-1
)
Fig. 4 DPPH radical scaveng-
ing activity expressed in oxida-
tion index EC
50
given in mg
mL
−1
(mean ± SD; n=3) for
Rhodophyta extracts and con-
trols (ascorbic acid, BHA, BHT
and α-tocopherol). Bars repre-
sent standard deviations. Signif-
icant differences are indicated
by different letters as deter-
mined by Tukey HSD test
(p<0.05)
454 J Appl Phycol (2007) 19:449–458
The most active species of Phaeophyta in our study
(Lobophora variegata,Padina gymnospora and Dictyota
cervicornis) belong to the same family, the Dictyotaceae,
which has been extensively studied for its wide variety of
bioactive compounds (e.g., terpenoids and acetogenins) that
conferred a very effective antiherbivore defense system and
various biological activities (Ballantine et al. 1987; Duran
et al. 1997; Amsler and Fairhead 2006). In previous studies,
low antioxidant activities have been measured in other
species of Dictyotaceae (Yan et al. 1998; Santoso et al.
2004; Cavas and Yurdakoc 2005), except in Spatoglossum
pacificum (Matsukawa et al. 1997).
The genus Sargassum also exhibited relatively high DPPH
radical scavenging activities with an oxidation index EC
50
rangingfrom6.64to7.14mgmL
−1
. The genus Sargassum has
been studied extensively showing high antioxidant potential
in vitro (Anggadiredja et al. 1997; Matsukawa et al. 1997;
Yan et al . 1998; Lim et al. 2002; Santoso et al. 2004;Heo
et al. 2005;Kimetal.2005;Parketal.2005; Connan et al.
2006) and in vivo (Mori et al. 2003; Wei et al. 2003).
Rhodophyta
All species of Rhodophyta showed antioxidant activities
with EC
50
from 2.84 to 77.71 mg mL
−1
(Fig. 4). Chondria
baileyana had the highest antioxidant activity with the
lowest EC
50
(2.84±0.07 mg mL
−1
) significantly equivalent
to EC
50
of all commercial antioxidants tested as well as the
highest phenolic content (7.30±0.29% dry wt) (Table 1).
Chondria atropurpurea also exhibited a relatively high
antioxidant activity (10.43±0.11 mg mL
−1
). In fact, the
genus Chondria is a source of various bioactive compounds
(e.g., terpenoids, novel amino-acids, cyclic polysulfides and
indoles) that have shown anthelmintic (Davyt et al. 1998)
and antibiotic activities (Ballantine et al. 1987). Chondria
crassicaulis and C. tenuissima have not shown a high
antioxidant activity (Huang and Wang 2004) when com-
pared to our results, but they were collected in temperate
environments.
Various species from the order Ceramiales also exhibited
relatively high DPPH radical scavenging activities: Hetero-
siphonia gibbesii (8.15±0.10 mg mL
−1
), Acanthophora
spicifera (12.50±0.24 mg mL
−1
), Bryothamnion triquetrum
(12.89±0.51 mg mL
−1
) and Ceramium nitens (13.89±
0.07 mg mL
−1
). Other studies have shown antioxidant
activity in Polysiphonia urceolata and P. morrowii (Fujimoto
1990), Bryothamnion triquetrum (Fallarero et al. 2003),
and Rhodomela confervoides (Huang and Wang 2004). On
the other hand, although the genus Laurencia and
Chondrophycus represent a prolific source of secondary
metabolites (Blunt et al. 2005), their antioxidant activity
was not comparable to other species from the order
Ceramiales. This observation is in accordance with
previous studies where Laurencia species have not shown
high antioxidant activity (Anggadiredja et al. 1997; Yan et al.
1998; Takamatsu et al. 2003; Kim et al. 2005). Gracilaria
species exhibited very low antioxidant activities (28.94 to
72.51 mg mL
−1
), although Yan et al. (1998)reportedaDPPH
radical scavenging activity around 40% for G. verrucosa
methanolic extract, though results are not comparable
because of the different methods used.
No discernable pattern of antioxidant activity could be
detected within a single algal order or division. It is difficult
to compare the antioxidant activity between all the above
species since they were collected at different periods and
localities. In fact, the production of antioxidant compounds,
like phenolics, is influenced by several factors: extrinsic
(herbivory pressure, irradiance, depth, salinity, nutrients,
etc.), and intrinsic (type, age and reproductive stage) (see
review in Connan et al. 2006). Therefore, antioxidant
activity of seaweeds could be subject to great intraspecific
variation, even at very small scales (Connan et al. 2006),
with intra-thallus variations in antioxidant activity having
been shown for Ascophyllum nodosum. Moreover, it is very
difficult to compare macroalgae antioxidant activities with
other studies because each author used different extraction
protocols, assay methods and units. This is why the use of
the oxidation index EC
50
represents a valuable tool for
comparisons.
Antioxidative activities of the most active species
Our study showed a very strong antioxidant potential in three
species of macroalgae: Lobophora variegata (0.32± 0.01 mg
mL
−1
), Avrainvillea longicaulis (1.44±0.01 mg mL
−1
)and
Chondria baileyana (2.84±0.07 mg mL
−1
). In fact, they are
equivalent to the activities of some commercial antioxidants.
Moreover, reducing activities of the extracts at two concen-
trations (0.1 and 0.5 mg mL
−1
) confirmed the results of the
DPPH method: L. variegata had the greatest reducing
activity, and its activity increased as extract concentration
increased (Fig. 5a). The highest levels of reducing activity
were measured for BHA, ascorbic acid and BHT, but the one
from L. variegata is significantly higher than those of
commercial antioxidant α-tocopherol. Most of the published
studies with macroalgae have demonstrated reducing activity
(Karawita et al. 2005;Kudaetal.2005;Yuanetal.2005;
Senevirathne et al. 2006).
Superoxide anion radical O
2
scavenging activity of
the extracts are summarized in Fig. 5b. Highest values were
for ascorbic acid and L. variegata (71.84% and 70.46% for
1mgmL
−1
, respectively) which confirm the strong
antioxidant activity of L. variegata as was the case with
various species of Phaeophyta (Siriwardhana et al. 2003;
Karawita et al. 2005; Kim et al. 2005; Kuda et al. 2005;
Senevirathne et al. 2006), but only Ecklonia cava compares
J Appl Phycol (2007) 19:449–458 455
with that found in L. variegata which may be due to its high
phenolic content. In this regard, Robak and Gryglewski
(1988) demonstrated that phenolic compounds are effective
mainly via the scavenging of superoxide anion which may
also explain the behaviour of A. longicaulis extract (19.71%
at 1 mg mL
−1
) presumably containing avrainvilleol (Sun et al.
1983). C. baileyana had very low superoxide anion radical
scavenging (5.91 and 9.08% at 0.1 and 0.5 mg mL
−1
,
respectively). Kim et al. (2005) reported very low superoxide
anion radical scavenging activities (< 5%) for various
extracts of Rhodophyta while higher values have been
reported only for Grateloupia filicina, ranging from 20 to
65% at 2 mg mL
−1
(Athukorala et al. 2003). In this study, C.
baileyana at 1 mg mL
−1
induced the production of superoxide
anion radical instead of inhibiting it (Fig. 5b). The pro-
oxidant activity of this extract could not be explained until
the antioxidant compound had been identified. However, it is
well know that some antioxidants,likeascorbicacidorα-
tocopherol, can act as pro-oxidants depending on dose and
experimental conditions (Zhang and Omaye 2001).
The inhibition of lipid peroxidation of seaweed extracts
is shown in Fig. 6. Auto-oxidation of linoleic acid without
0.0
0.4
0.8
1.2
1.6
0246810
Incubation
p
eriod
(
da
y
s
)
Inhibition of lipid oxidation (Abs 500 nm)
Control
BHA (c)
BHT (a)
-tocopherol (d)
Avrainvil lea longi cauli s (b)
Chondria bai leyana (a)
Lobophora variegat a (d)
Fig. 6 Inhibition of linoleic acid oxidation by A. longicaulis,C.
baileyana and L. variegata extracts and commercial antioxidants
(BHA, BHT and α-tocopherol) at a concentration of 0.5 mg mL
−1
.
Bars represent standard deviation. Significant differences are indicated
in the legend by different letters as determined by Tukey HSD test (p<
0.05)
a
b
c
d
e
f
f
0
1
2
3
Avrainvillea
longicaulis
Chondria
baileyana
Lobo pho ra
variegat a
Ascorbic
acid
BHA BHT -tocopherol
Reducing activity (Abs 700 nm)
0.1 mg mL
0.5 mg mL
aa
b
c
c
d
-10
10
30
50
70
Avrainvillea
lon
g
icaulis
Chondria
baile
y
ana
Lobophora
varie
g
ata
Ascorbic acid BHT -tocopherol
O2
-
scavenging activity (%)
0.1mg mL
0.5 mg mL
1 mg mL
-1
-1
-1
-1
-1
a
b
Fig. 5 Antioxidant activity of
A. longicaulis,C. baileyana and
L. variegata extracts compared
to commercial antioxidants
(ascorbic acid, BHA, BHT and
α-tocopherol). aReducing ac-
tivity (absorbance at 700 nm); b
superoxide anion radical scav-
enging activity (%). Bars repre-
sent standard deviations.
Significant differences are indi-
cated by different letters as
determined by Tukey HSD test
(p<0.05)
456 J Appl Phycol (2007) 19:449–458
the addition of algal extracts or commercial antioxidants
was accompanied by a rapid increased in absorbance that
reached 1.540 in 10 days (control). The inhibitory effect of
C. baileyana at 0.5 mg mL
−1
is equivalent to BHT and
significantly higher than the effects of commercial antiox-
idants tested. A. longicaulis also showed inhibitory effect of
lipid peroxidation, significantly higher than BHA and α-
tocopherol. In this assay, the antioxidant activity of L.
variegata showed the lowest antioxidant activity; neverthe-
less, its inhibitory effect is equivalent to that of α-tocopherol.
These data are in accordance with previous studies that have
demonstrated the inhibition of lipid peroxidation by extracts
from Rhodophyta (Athukorala et al. 2003;Yuanetal.2005),
Phaeophyta (Lim et al. 2002;Siriwardhanaetal.2003;
Karawita et al. 2005; Senevirathne et al. 2006)and
Chlorophyta (Cavas and Yurdakoc 2005). Moreover, these
results suggest that antioxidants from C. baileyana and A.
longicaulis are more effective as chain breaking molecules
rather than reductors, whereas those of L. variegata have
very good reductive capacity, but low chain breaking
capacity. In the future, identification of these molecules will
be helpful to understand the different antioxidant mecha-
nisms observed in this study.
Conclusion
This work represents the largest screening of antioxidant
activity in tropical macroalgae to date. All species collected
from the coasts of Quintana Roo and Yucatan showed
antioxidant activities, suggesting that tropical macroalgae
develop an effective antioxidant defense system which may
reflect an adaptation to high solar radiation. This screening
emphasized the great antioxidant potential (free-radical,
superoxide anion radical scavenging, reducing activity, and
inhibition of lipid peroxidation) of three species: Lobophora
variegata,Avrainvillea longicaulis and Chondria baileyana.
Identification of the antioxidant compounds of these extracts
will lead to their evaluation in medicine, food production
and cosmetic industry.
Acknowledgments This research was financed by SAGARPA-
CONACYT (Contract 2002-C01-1057). The authors thank J.L. Godinez
for identification of the macroalgae species and C. Chávez and M.L.
Zaldivar for technical assistance.
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