Journal of Applied Phycology 13: 403407, 2001.

2001 Kluwer Academic Publishers. Printed in the Netherlands.

403

Antioxidant activity of extracts of the marine algal genus Cystoseira in a

micellar model system

Giuseppe Ruberto1 , Maria T. Baratta1 , Daniela M. Biondi1 & Vincenzo Amico2

1 Istituto
del C.N.R. per lo Studio delle Sostanze Naturali di Interesse Alimentare e Chimico-Farmaceutico# Via del
Santuario 110, I-95028 Valverde (CT), Italy
2 Dipartimento di Scienze Chimiche, Universita degli Studi, Viale A. Doria 6, I-95125 Catania, Italy
# Associated to the National Institute for the Chemistry of Biological Systems C.N.R.

( Author for correspondence; phone +39 0957212136; fax +39 0957212141; e-mail ruberto@issn.ct.cnr.it)

Received 15 October 2000; revised 7 December 2000; accepted 7 December 2000

Key words: antioxidants, Cystoseira species, marine algae, relative antioxidant efficiency (RAE), -tocopherol,
tetraprenyltoluquinols

Abstract
The antioxidant activity of the lipid extracts of eight marine algae belonging to the Cystoseira genus has been
evaluated in a micellar model system. The activity has been ascribed to the presence in the extracts of tetrapren-
yltoluquinols, which are tocopherol-like compounds characteristic of these algae. Results have been expressed as
relative antioxidant efficiency (RAE), defined as the ratio of the antioxidant efficiency (AE) of the tested extract to
that of -tocopherol. From the results a composition-activity relationship has been deduced.

Introduction ins and/or important lipids. For these reasons many

The effects of light, singlet oxygen and active free rad-
icals (O2 , OH , LOO ) on living systems are ever
more the object of research (Barclay, 1993; Breccia et
al., 1986; Girotti, 1990; Stadtman, 1992; Sun, 1990).
DNA, cell membranes, proteins and other cellular con-
stituents are the target molecules of the degradation
processes, and their modifications represent the basis
of several pathological disorders, such as atheroscler-
osis, rheumatoid arthritis, muscular dystrophy, catar-
acts, some neurological disorders and some types of
cancer. Furthermore the radical reactions are strongly
involved in the ageing processes (Steinberg et al.,
1989; Gordon, 1996).
Another field strongly affected by the same species
with as many detrimental effects is the food sector.
It is well known, in fact, that the radical peroxida-
tion of lipids, contained in almost all foods, is the
predominant cause of qualitative decay of foods, both
during their storage and transformations (St. Angelo,
1992). The main effects are bad-smelling substances,
in some cases toxic, and the destruction of vitam-
products with antioxidant properties are widely used
to increase the shelf life of foodstuffs; they are mainly
of synthetic origin and, chemically, are phenol deriv-
atives. Nevertheless some of these compounds, such
as butylhydroxytoluene (BHT) and butylhydroxyan-
isole (BHA), have recently been suspected of toxicity
(Madsen & Bertelsen, 1995; Safer & Al-Nughamish,
1999). Therefore attention is focusing on the develop-
ment of new, safe and cheap antioxidants, possibly of
natural origin (Gordon, 1996; Larson, 1988; Lewis,
1989). Higher plants and their constituents are, in fact,
continuously being investigated for their potential an-
tioxidant effectiveness (Fukumoto & Mazza, 2000);
more recently several screening studies on marine al-
gae showed that a similar activity may be found in
several seaweeds (Anggardiredja et al., 1997; Mat-
sukawa et al., 1997; Tutour et al., 1998; Yan et al.,
1999).
Phenols are particularly effective antioxidants for
polyunsaturated fatty acids; in fact they easily transfer
a hydrogen atom to lipid peroxyl radicals (LOO ), and
form the aryloxyl radical (ArO), which being incap-
404
able of acting as a chain carrier, couples with another
radical thus quenching the radical process (Scheme 1).
LOO + ArOH LOOH + ArO
ArO + LOO non-radical products
ArO + ArO non-radical products
Scheme 1 Quencing reactions of a radical process.
Phenolic compounds are present throughout the plant
kingdom (Larson, 1988) and among them, the tet-
raprenyltoluquinols, namely compounds formed by
the coupling of a hydroquinone ring and a diterpen-
oidic chain, are characteristic secondary metabolites
of the genus Cystoseira (Amico, 1995; Piattelli, 1990).
Considering that tocopherols, the most important nat-
ural antioxidants, are tetraprenyltoluquinols, as well
as the fact that some of these algal metabolites, previ-
ously tested as antioxidants, gave results comparable
to that of -tocopherol (Foti et al., 1994), we were
prompted to assay the antioxidant activity of the lipid
extract of these algae in a micellar model system (Foti
et al., 1996; Pryor et al., 1993).
Species of Cystoseira are the most widespread
marine flora along the Mediterranean coasts and rep-
resent one of the most important elements of the
ecosystem. The genus is also one of the most studied
marine genera, from both from biological and chem-
ical viewpoints, and has been used as a model system
for chemotaxonomic studies (Amico, 1995).
The phytochemical peculiarity of this genus is its
synthesis and accumulation of secondary metabolites
of mixed biogenesis, namely tetraprenyltoluquinols in
which the benzene ring presents a 1,4 dihydroxy, a
6 methyl and a 2 diterpenoid chain substitution. The
differentiating element among the several species of
Cystoseira is the diterpenoid chain that in some cases
is very simple, being linear and little functionalised,
whereas in other cases it is very complex being largely
cyclised and functionalised (Amico, 1995).
The aim of this study was to characterize the
antioxidant activity of the lipid extracts of eight Cys-
toseira taxa: C. amentacea var. stricta, C. amentacea
var. amentacea, C. algeriensis, C. elegans, C. al-
geriensis C. elegans, C. jabukae, C. barbata and
C. crinita, collected along the Sicilian coasts. The
antioxidant effectiveness, considered as the capacity
to protect linoleic acid subjected to peroxidation in
aqueous micelles of sodium dodecyl sulphate (SDS)
in buffer solution at pH 7.4 and 50 C, was meas-
ured. The results are expressed as relative antioxidant
efficiency (RAE), defined as the ratio of the antiox-
idant efficiency (AE) of the tested extracts to that of
-tocopherol (Pryor et al., 1993).
Materials and methods
General experimental procedure
Linoleic acid and SDS were purchased from Sigma,
2,2 -azobis(2-amidinopropano)dihydrochloride was
from Wako, and -tocopherol from Aldrich. All were
used without further purification. Kinetics were recor-
ded on a Beckman DU-65 spectrophotometer with a
thermostated cuvette house and a PC to process the
data.
Plant material
Algae were collected in July 1998 along the coasts of
south-eastern Sicily. Cystoseira amentacea Bory var.
amentacea Bory, C. jabukae Ercegovic and C. crinita
(Desf.) Bory were collected at Castelluccio (Siracusa);
C. amentacea Bory var. stricta Montagne, C. elegans
Sauv., C. algeriensis J. Feldm., C. elegans C. al-
geriensis Giaccone and C. barbata C. Agardh were
collected at Portopalo (Siracusa). Voucher specimens
are deposited at the Herbarium of the Department of
Botany, Catania, Italy.
Extraction
Plant material was freeze-dried, ground and extrac-
ted three times with CH2 Cl2 at room temperature
with continuous stirring. The extracts were pooled
and evaporated at reduced pressure to give green oils
which were stored under N2 at 0 C until used. 100 g
dried material gave the following yields of organic ex-
tracts: 0.67 g (C. amentacea var. amentacea), 0.43 g
(C. jabukae), 0.31 g (C. crinita), 0.89 g (C. amentacea
var. stricta), 0.35 g (C. elegans), 0.52 g (C. algerien-
sis), 0.21 g (C. elegans C. algeriensis), 0.37 g (C.
barbata).
Determination of antioxidant activity
Solutions
A 0.1 M solution of SDS was prepared in aqueous
0.01 M NaH2 PO4 and adjusted to pH 7.4 with con-
centrated aqueous NaOH. Linoleic acid was added,
immediately before each experiment, to a concen-
tration of 0.0026 M. A stock solution (0.07 M) of
 405

2,2 -azobis(amidinopropane)dihydrochloride in water,

stored at 510 C, was used within a week. Solutions
of extracts to be tested (1520 mg L1 ) in methanol
were prepared immediately before use.

Procedure

An aliquot (2 mL) of the micellar suspension of li-

noleic acid and scalar amounts of antioxidant solution
was stirred and kept at 50 C for 20 minutes in the
sample compartment of the spectrophotometer. The
buffered SDS solution was used as blank.The progress
of the peroxidation of the linoleic acid was monitored
for 15 minutes recording the absorbance at 234 nm
after addition of 10 L radical initiator solution. The
slope of the linear plot of absorbance vs. time gives
dA/dt. From the plots dA/dt vs. [InH]1 the slope Sinh
was obtained and the RAE values calculated.
Figure 1. Calculation of AE values for:
-tocopherol:  C.
amentacea var. stricta;
C. elegans;  C. jabukae extracts in
0.10 M SDS / 0.05 M phosphate buffer (pH 7.4), at 50 C (SD =
12%).
Table 1. Relative antioxidant efficiency (RAE) values of Cysto-
seira extracts (n = 4; SD = 15%). For experimental protocols,
see Materials and methods
Species RAE

-tocopherol 1.00
Results Cystoseira amentacea var. stricta 0.83
Cystoseira amentacea var. amentacea 0.57
The model system to assay the antioxidant activity is Cystoseira algeriensis 0.54
based on the spectrophotometric determination of the Cystoseira elegans 0.62
Cystsoeira elegans C. algeriensis 0.31
rate of conjugated diene formation from LH (linoleic
Cystoseira jabukae 0.37
acid), in the presence of either -tocopherol or a po-
Cystoseira barbata 0.58
tential antioxidant. Conjugated diene hydroperoxides
Cystoseira crinita 0.43
(LOOH) are the end products of LH peroxidation in-
duced into the micellar phase by a radical initiator
which, on thermolysis, provides radicals at a constant
rate. These hydroperoxides have strong UV absorp-
tion, in micelles of SDS in buffer solution at pH and inhibition, respectively. The two competitive reactions in the
7.4, with a maximum at 234 nm and a molar coeffi- model system are in bold.
cient of 26,100 M1 cm1 . Scheme 2 summarises the
reactions involved. The kinetic treatment of the method correlates the
 absorbance variation (dA/dt) with the concentration of
R-N=N-R 2R + N2 antioxidant ([InH]1). Plotting these two values gives
R + O2 ROO a straight line with the slope S. The ratio between
ROO + LH ROOH + L Stoc and Sinh gives the RAE values for the tested
L + O2 LOO inhibitor. Figure 1 illustrates typical examples of the
kp plot dA/dt vs. [InH]1 , while Table 1 shows the res-
LOO + LH LOOH + L ults of the antioxidant activity of algal extracts in the
kt
2LOO non-radical products micellar system.
kinh
LOO + InH LOOH + In
LOO + In non-radical products
Discussion

Scheme 2 Reactions involved in the micellar model system. R-N=N- Figure 2 shows some tetraprenyltoluquinols isolated
R is the radical initiator, LH is linoleic acid, L a linoleic radical, from Mediterranean Cystoseira taxa in this study.
LOO a linoleic peroxyl radical, and InH the antioxidant (inhibitor); C. amentacea var. stricta extract is the most active
kp , kt , and kinh are rate constants of the propagation, termination, having a RAE of 0.83 (Table 1). Among those studied,
406
Figure 2. Selected tetraprenyltoluquinols from Mediterranean Cystoseira species.
this alga is one of the richest in tetraprenyltoluquinols
with an intrinsic capability of transferring a hydrogen
atom to the peroxyl radical LOO . These metabol-
ites, an example of which is given by cystoketal
and strictaketal metabolites (1, 2; Figure 2), in fact,
amount to 12% ca. of the organic extract, among the
highest values for this genus (Amico et al., 1987).
Similar metabolites are present in the extract of C.
amentacea var. amentacea (3, 4; Figure 2), but the
value of RAE is 0.57 (Table 1). This behaviour is to be
ascribed to a lower concentration of these metabolites,
half that of the previous alga (Amico et al., 1986).
The RAE values of the C. algeriensis, C. eleg-
ans and their natural hybrid extracts are 0.54, 0.62
and 0.31, respectively. When the above data are con-
sidered together with the composition of the lipid
extract some discrepancy appears. In fact, tetrapren-
yltoluquinols of these algae are mainly present with
both phenolic functions methylated, and thus unsuit-
able for antioxidant activity, whereas those with free
phenols are present to a lower extent (Figure 2). In
particular, C. algeriensis shows exclusively tetrapren-
yltoluquinols with a bicyclic diterpenic moiety (5, 6);
C. elegans accumulates metabolites chemically cor-
related to the previous ones, but with a monocyclic
diterpenic moiety (7, 8); finally the natural hybrid
shows the presence of all the compounds (Amico et
al., 1988a). This anomalous behaviour can be ascribed
to a higher capability of these metabolites, with re-
spect to the previous ones, of transferring the hydrogen
atom, and also to a higher degree of lipophilicity of the
extract of these algae, which would ensure a greater
amount of these compounds inside the micelles where
all the reactions take place.
The metabolites of C. jabukae extract are repres-
ented by a hydroquinol and a monomethyl derivatives,
namely compounds 9 and 10 of Figure 2, and two
tetraprenyltoluquinones (Amico et al., 1985). In this
case it is clear that the scarce presence of phenolic
hydrogen atoms is correlated to the low antioxidant
activity (RAE of 0.37) (Table 1).
The RAE of C. barbata extract (0.58) is relatively
high in relation to its low tetraprenyltoluquinols con-
tent. Only one metabolite (11; Figure 2) of mixed
biogenesis is present, but the presence of 0.12% -
tocopherol in the extract must be taken into account
(Amico et al., 1990). This is a rare example in which
the presence of this compound is at the same level
as the other secondary metabolites; its presence and
a probable synergistic effect must be considered to

explain the high RAE value. The remaining alga, C.
crinita, has a lipid extract with the same compound 11
and the corresponding quinone (Amico et al., 1988b):
-tocopherol is absent and the RAE is 0.43 (Table1).
This study confirms the potential of the micellar
system in the rapid determination of antioxidant activ-
ity of not only of pure compounds, but also total
extracts endowed with a degree of lipophilicity. The
lipid extracts of Cystoseira show interesting activities
in the model system, the observed differences being
ascribable to the differences in tetraprenyltoluquinols
content in the extracts. Therefore, this marine genus
could represent a new and alternative source of antiox-
idant components suitable for exploitation in the food
and cosmetic sectors as shelf-life prolonging additives.
Acknowledgements
We thank Mr A Renda (ISSN-CNR, Valverde) and Dr
L Siracusa for skilful technical assistance. This work
was supported by Consiglio Nazionale delle Ricerche
(C.N.R. Rome) and by a MURST 40% grant.
References
Amico V (1995) Marine brown algae of family of Cystoseiraceae:
Chemistry and chemotaxonomy. Phytochemistry 39(6): 1257
1279.
Amico V, Cunsolo F, Piattelli M, Ruberto G (1985) Tetraprenyltolu-
quinols from the brown alga Cystoseira jabukae. Phytochemistry
24(5): 10471050.
Amico V, Cunsolo F, Piattelli M, Ruberto G, Mayol L (1987)
Strictaketal, a new tetraprenyltoluquinol with a heterotetracyclic
diterpene moiety from the brown alga Cystoseira stricta. J. nat.
Prod. 50(3): 449454.
Amico V, Giaccone G, Piattelli M, Ruberto G (1988a) Inherit-
ance of chemical constituents in algae: Tetraprenyltoluquinols
of Cystoseira elegans C. algeriensis. Phytochemistry 27(4):
10691071.
Amico V. Piattelli M, Cunsolo F, Recupero M, Ruberto G (1990)
Tetraprenyltoluquinols as chemotaxonomic markers in the genus
Cystoseira: C. barbatula and C. barbata. Gazz. Chim. Ital. 120:
912.
Amico V, Piattelli M, Neri P, Ruberto G (1988b) Meroterpenoids
from Cystoseira spp. J. Nat. Prod. 51(1): 191192.
Amico V, Piattelli M, Neri P, Ruberto G, Mayol L (1986) Novel
metabolites from the marine genus Cystoseira Application of
two-dimensional 1 H13 C correlation to the structure elucida-
tion. Tetrahedron 42(21): 60156020.
Anggardiredja J, Andyani R, Muaswanah H (1997) Antioxidant
activity of Sargassum polycystum and Laurencia obtusa from
Scribu Islands. J. appl. Phycol. 9: 477479.
Barclay LRC (1993) Model biomembrane: quantitative studies
of peroxidation, antioxidant action, partitioning, and oxidative
stress. Can. J. Chem. 71: 116.
407
Breccia A, Rodgers MAJ, Semeraro G (1986) Oxygen and Sulfur
Radicals in Chemistry and medicine, Oxygen Radicals in Chem-
istry and Biology (Chap.1); Edizioni Scientifiche Lo Scarabeo,
Bologna, pp. 2052.
Foti M, Piattelli M, Amico V, Ruberto G (1994) Antioxidant activity
of phenolic meroditerpenoids from marine algae. J. Photochem.
Photobiol. B 26: 159164.
Foti M, Piattelli M, Baratta MT, Ruberto G (1996) Flavonoids,
coumarins, and cinnamic acids as antioxidants in a micellar
system. Structure-activity relationship. J. agric. Food Chem. 44:
497501.
Fukumoto LR, Mazza G (2000) Assessing antioxidant and proox-
idant activities of phenolic compounds. J. agric Food Chem. 48:
35973604.
Girotti AW (1990) Photodynamic peroxidation in biological sys-
tems. Photochem. Photobiol. 51: 497509.
Gordon MH (1996) Dietary antioxidants in disease prevention. Nat.
Prod. Rep. 13: 265273.
Halliwell B (1987) Oxidant and human disease: some new concepts.
FASEB J. 1: 358364.
Larson RA (1988) The antioxidants from higher plants. Phytochem-
istry 27: 969978.
Lewis DA (1989) Anti-inflammatory Drugs from Plant and Marine
Source, Birkuser-Verlag, Basel, pp. 95283.
Madsen HL, Bertelsen G (1995) Spices as antioxidants. Trends Food
Sci. Technol. 6: 271277.
Matsukawa R, Dubinsky Z, Kishimoto E, Masaki K, Masuda Y,
Takeuchi T, Chihara M, Yamamoto Y, Niki E, Karube I (1997)
A comparison of screening methods for antioxidant activity in
seaweeds. J. appl. Phycol. 9: 2935.
Piattelli M (1990) Chemistry and taxonomy of Sicilian Cystoseira
species. New J. Chem. 14: 777782.
Pryor WA, Cornicelli JA, Devall LJ, Tait B, Trivedi BK, Witiak DT,
Wu M (1993) A rapid screening test to determine the antioxidant
potencies of natural and synthetic antioxidants. J. org. Chem 58:
35213532.
Safer AM, Al-Nughamish (1999) Hepatotoxicity induced by the
antioxidant food additive butylated hydroxytoluene (BHT) in
rats: An electron microscopical study. Histol. Histopathol. 14:
391406.
Sdatman E R (1992) Protein oxidation and ageing. Science 257:
12201224.
St. Angelo AJ (1992) Lipid Oxidation in Food. ACS Symposium
Series 500, Washington DC.
Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum J
(1989) Modification of low density lipoprotein that increase its
atherogenicity. New Engl. J. Med. 320: 915924
Sun Y (1990) Free radicals, antioxidant enzymes, and carcinogen-
esis. Free Radicals Biol. Med. 8: 583599.
Tutour BI, Benslimane F, Gouleau MP, Gouygou JP, Saadan B,
Quemeneur F (1998) Antioxidant and pro-oxidant activities of
brown algae, Laminaria digitata, Himanthalia elongata, Fucus
vesiculosus, Fucus serratus and Ascophyllum nodosum. J. appl.
Phycol. 10: 121129.
Yan X, Chuda Y, Suzuki M, Nagata T (1999) Fucoxanthin as the ma-
jor antioxidant in Hijikia fusiformis, a common edible seaweed.
Biosci. Biotechnol. Biochem. 63: 605607.
Yen GC, Duh PD, Tsai CL (1993) Relationship between antioxidant
activity and maturity of peanut hulls. J. agric. Food Chem. 41:
6770.