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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


products with antioxidant properties are widely used
The effects of light, singlet oxygen and active free rad- to increase the shelf life of foodstuffs; they are mainly
icals (O2 , OH , LOO ) on living systems are ever of synthetic origin and, chemically, are phenol deriv-
more the object of research (Barclay, 1993; Breccia et atives. Nevertheless some of these compounds, such
al., 1986; Girotti, 1990; Stadtman, 1992; Sun, 1990). as butylhydroxytoluene (BHT) and butylhydroxyan-
DNA, cell membranes, proteins and other cellular con- isole (BHA), have recently been suspected of toxicity
stituents are the target molecules of the degradation (Madsen & Bertelsen, 1995; Safer & Al-Nughamish,
processes, and their modifications represent the basis 1999). Therefore attention is focusing on the develop-
of several pathological disorders, such as atheroscler- ment of new, safe and cheap antioxidants, possibly of
osis, rheumatoid arthritis, muscular dystrophy, catar- natural origin (Gordon, 1996; Larson, 1988; Lewis,
acts, some neurological disorders and some types of 1989). Higher plants and their constituents are, in fact,
cancer. Furthermore the radical reactions are strongly continuously being investigated for their potential an-
involved in the ageing processes (Steinberg et al., tioxidant effectiveness (Fukumoto & Mazza, 2000);
1989; Gordon, 1996). more recently several screening studies on marine al-
Another field strongly affected by the same species gae showed that a similar activity may be found in
with as many detrimental effects is the food sector. several seaweeds (Anggardiredja et al., 1997; Mat-
It is well known, in fact, that the radical peroxida- sukawa et al., 1997; Tutour et al., 1998; Yan et al.,
tion of lipids, contained in almost all foods, is the 1999).
predominant cause of qualitative decay of foods, both Phenols are particularly effective antioxidants for
during their storage and transformations (St. Angelo, polyunsaturated fatty acids; in fact they easily transfer
1992). The main effects are bad-smelling substances, a hydrogen atom to lipid peroxyl radicals (LOO ), and
in some cases toxic, and the destruction of vitam- form the aryloxyl radical (ArO), which being incap-
404

able of acting as a chain carrier, couples with another idant efficiency (AE) of the tested extracts to that of
radical thus quenching the radical process (Scheme 1). -tocopherol (Pryor et al., 1993).
LOO + ArOH LOOH + ArO
ArO + LOO non-radical products
Materials and methods
ArO + ArO non-radical products
General experimental procedure
Scheme 1 Quencing reactions of a radical process.

Phenolic compounds are present throughout the plant Linoleic acid and SDS were purchased from Sigma,
kingdom (Larson, 1988) and among them, the tet- 2,2 -azobis(2-amidinopropano)dihydrochloride was
raprenyltoluquinols, namely compounds formed by from Wako, and -tocopherol from Aldrich. All were
the coupling of a hydroquinone ring and a diterpen- used without further purification. Kinetics were recor-
oidic chain, are characteristic secondary metabolites ded on a Beckman DU-65 spectrophotometer with a
of the genus Cystoseira (Amico, 1995; Piattelli, 1990). thermostated cuvette house and a PC to process the
Considering that tocopherols, the most important nat- data.
ural antioxidants, are tetraprenyltoluquinols, as well
as the fact that some of these algal metabolites, previ- Plant material
ously tested as antioxidants, gave results comparable
Algae were collected in July 1998 along the coasts of
to that of -tocopherol (Foti et al., 1994), we were
south-eastern Sicily. Cystoseira amentacea Bory var.
prompted to assay the antioxidant activity of the lipid
amentacea Bory, C. jabukae Ercegovic and C. crinita
extract of these algae in a micellar model system (Foti
(Desf.) Bory were collected at Castelluccio (Siracusa);
et al., 1996; Pryor et al., 1993).
C. amentacea Bory var. stricta Montagne, C. elegans
Species of Cystoseira are the most widespread
Sauv., C. algeriensis J. Feldm., C. elegans C. al-
marine flora along the Mediterranean coasts and rep-
geriensis Giaccone and C. barbata C. Agardh were
resent one of the most important elements of the
collected at Portopalo (Siracusa). Voucher specimens
ecosystem. The genus is also one of the most studied
are deposited at the Herbarium of the Department of
marine genera, from both from biological and chem-
Botany, Catania, Italy.
ical viewpoints, and has been used as a model system
for chemotaxonomic studies (Amico, 1995).
Extraction
The phytochemical peculiarity of this genus is its
synthesis and accumulation of secondary metabolites Plant material was freeze-dried, ground and extrac-
of mixed biogenesis, namely tetraprenyltoluquinols in ted three times with CH2 Cl2 at room temperature
which the benzene ring presents a 1,4 dihydroxy, a with continuous stirring. The extracts were pooled
6 methyl and a 2 diterpenoid chain substitution. The and evaporated at reduced pressure to give green oils
differentiating element among the several species of which were stored under N2 at 0 C until used. 100 g
Cystoseira is the diterpenoid chain that in some cases dried material gave the following yields of organic ex-
is very simple, being linear and little functionalised, tracts: 0.67 g (C. amentacea var. amentacea), 0.43 g
whereas in other cases it is very complex being largely (C. jabukae), 0.31 g (C. crinita), 0.89 g (C. amentacea
cyclised and functionalised (Amico, 1995). var. stricta), 0.35 g (C. elegans), 0.52 g (C. algerien-
The aim of this study was to characterize the sis), 0.21 g (C. elegans C. algeriensis), 0.37 g (C.
antioxidant activity of the lipid extracts of eight Cys- barbata).
toseira taxa: C. amentacea var. stricta, C. amentacea
var. amentacea, C. algeriensis, C. elegans, C. al- Determination of antioxidant activity
geriensis C. elegans, C. jabukae, C. barbata and
C. crinita, collected along the Sicilian coasts. The Solutions
antioxidant effectiveness, considered as the capacity
to protect linoleic acid subjected to peroxidation in A 0.1 M solution of SDS was prepared in aqueous
aqueous micelles of sodium dodecyl sulphate (SDS) 0.01 M NaH2 PO4 and adjusted to pH 7.4 with con-
in buffer solution at pH 7.4 and 50 C, was meas- centrated aqueous NaOH. Linoleic acid was added,
ured. The results are expressed as relative antioxidant immediately before each experiment, to a concen-
efficiency (RAE), defined as the ratio of the antiox- tration of 0.0026 M. A stock solution (0.07 M) of
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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 Figure 1. Calculation of AE values for:  -tocopherol:  C.
buffered SDS solution was used as blank.The progress amentacea var. stricta;  C. elegans;  C. jabukae extracts in
of the peroxidation of the linoleic acid was monitored 0.10 M SDS / 0.05 M phosphate buffer (pH 7.4), at 50 C (SD =
for 15 minutes recording the absorbance at 234 nm 12%).
after addition of 10 L radical initiator solution. The Table 1. Relative antioxidant efficiency (RAE) values of Cysto-
slope of the linear plot of absorbance vs. time gives seira extracts (n = 4; SD = 15%). For experimental protocols,
dA/dt. From the plots dA/dt vs. [InH]1 the slope Sinh see Materials and methods
was obtained and the RAE values calculated.
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 diterpenic moiety (7, 8); finally the natural hybrid
with an intrinsic capability of transferring a hydrogen shows the presence of all the compounds (Amico et
atom to the peroxyl radical LOO . These metabol- al., 1988a). This anomalous behaviour can be ascribed
ites, an example of which is given by cystoketal to a higher capability of these metabolites, with re-
and strictaketal metabolites (1, 2; Figure 2), in fact, spect to the previous ones, of transferring the hydrogen
amount to 12% ca. of the organic extract, among the atom, and also to a higher degree of lipophilicity of the
highest values for this genus (Amico et al., 1987). extract of these algae, which would ensure a greater
Similar metabolites are present in the extract of C. amount of these compounds inside the micelles where
amentacea var. amentacea (3, 4; Figure 2), but the all the reactions take place.
value of RAE is 0.57 (Table 1). This behaviour is to be The metabolites of C. jabukae extract are repres-
ascribed to a lower concentration of these metabolites, ented by a hydroquinol and a monomethyl derivatives,
half that of the previous alga (Amico et al., 1986). namely compounds 9 and 10 of Figure 2, and two
The RAE values of the C. algeriensis, C. eleg- tetraprenyltoluquinones (Amico et al., 1985). In this
ans and their natural hybrid extracts are 0.54, 0.62 case it is clear that the scarce presence of phenolic
and 0.31, respectively. When the above data are con- hydrogen atoms is correlated to the low antioxidant
sidered together with the composition of the lipid activity (RAE of 0.37) (Table 1).
extract some discrepancy appears. In fact, tetrapren- The RAE of C. barbata extract (0.58) is relatively
yltoluquinols of these algae are mainly present with high in relation to its low tetraprenyltoluquinols con-
both phenolic functions methylated, and thus unsuit- tent. Only one metabolite (11; Figure 2) of mixed
able for antioxidant activity, whereas those with free biogenesis is present, but the presence of 0.12% -
phenols are present to a lower extent (Figure 2). In tocopherol in the extract must be taken into account
particular, C. algeriensis shows exclusively tetrapren- (Amico et al., 1990). This is a rare example in which
yltoluquinols with a bicyclic diterpenic moiety (5, 6); the presence of this compound is at the same level
C. elegans accumulates metabolites chemically cor- as the other secondary metabolites; its presence and
related to the previous ones, but with a monocyclic a probable synergistic effect must be considered to
407

explain the high RAE value. The remaining alga, C. Breccia A, Rodgers MAJ, Semeraro G (1986) Oxygen and Sulfur
crinita, has a lipid extract with the same compound 11 Radicals in Chemistry and medicine, Oxygen Radicals in Chem-
istry and Biology (Chap.1); Edizioni Scientifiche Lo Scarabeo,
and the corresponding quinone (Amico et al., 1988b): Bologna, pp. 2052.
-tocopherol is absent and the RAE is 0.43 (Table1). Foti M, Piattelli M, Amico V, Ruberto G (1994) Antioxidant activity
This study confirms the potential of the micellar of phenolic meroditerpenoids from marine algae. J. Photochem.
system in the rapid determination of antioxidant activ- Photobiol. B 26: 159164.
Foti M, Piattelli M, Baratta MT, Ruberto G (1996) Flavonoids,
ity of not only of pure compounds, but also total coumarins, and cinnamic acids as antioxidants in a micellar
extracts endowed with a degree of lipophilicity. The system. Structure-activity relationship. J. agric. Food Chem. 44:
lipid extracts of Cystoseira show interesting activities 497501.
in the model system, the observed differences being Fukumoto LR, Mazza G (2000) Assessing antioxidant and proox-
idant activities of phenolic compounds. J. agric Food Chem. 48:
ascribable to the differences in tetraprenyltoluquinols 35973604.
content in the extracts. Therefore, this marine genus Girotti AW (1990) Photodynamic peroxidation in biological sys-
could represent a new and alternative source of antiox- tems. Photochem. Photobiol. 51: 497509.
idant components suitable for exploitation in the food Gordon MH (1996) Dietary antioxidants in disease prevention. Nat.
Prod. Rep. 13: 265273.
and cosmetic sectors as shelf-life prolonging additives. 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.
Acknowledgements
Lewis DA (1989) Anti-inflammatory Drugs from Plant and Marine
Source, Birkuser-Verlag, Basel, pp. 95283.
We thank Mr A Renda (ISSN-CNR, Valverde) and Dr Madsen HL, Bertelsen G (1995) Spices as antioxidants. Trends Food
L Siracusa for skilful technical assistance. This work Sci. Technol. 6: 271277.
Matsukawa R, Dubinsky Z, Kishimoto E, Masaki K, Masuda Y,
was supported by Consiglio Nazionale delle Ricerche Takeuchi T, Chihara M, Yamamoto Y, Niki E, Karube I (1997)
(C.N.R. Rome) and by a MURST 40% grant. 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.
References Pryor WA, Cornicelli JA, Devall LJ, Tait B, Trivedi BK, Witiak DT,
Wu M (1993) A rapid screening test to determine the antioxidant
Amico V (1995) Marine brown algae of family of Cystoseiraceae: potencies of natural and synthetic antioxidants. J. org. Chem 58:
Chemistry and chemotaxonomy. Phytochemistry 39(6): 1257 35213532.
1279. Safer AM, Al-Nughamish (1999) Hepatotoxicity induced by the
Amico V, Cunsolo F, Piattelli M, Ruberto G (1985) Tetraprenyltolu- antioxidant food additive butylated hydroxytoluene (BHT) in
quinols from the brown alga Cystoseira jabukae. Phytochemistry rats: An electron microscopical study. Histol. Histopathol. 14:
24(5): 10471050. 391406.
Amico V, Cunsolo F, Piattelli M, Ruberto G, Mayol L (1987) Sdatman E R (1992) Protein oxidation and ageing. Science 257:
Strictaketal, a new tetraprenyltoluquinol with a heterotetracyclic 12201224.
diterpene moiety from the brown alga Cystoseira stricta. J. nat. St. Angelo AJ (1992) Lipid Oxidation in Food. ACS Symposium
Prod. 50(3): 449454. Series 500, Washington DC.
Amico V, Giaccone G, Piattelli M, Ruberto G (1988a) Inherit- Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum J
ance of chemical constituents in algae: Tetraprenyltoluquinols (1989) Modification of low density lipoprotein that increase its
of Cystoseira elegans C. algeriensis. Phytochemistry 27(4): atherogenicity. New Engl. J. Med. 320: 915924
10691071. Sun Y (1990) Free radicals, antioxidant enzymes, and carcinogen-
Amico V. Piattelli M, Cunsolo F, Recupero M, Ruberto G (1990) esis. Free Radicals Biol. Med. 8: 583599.
Tetraprenyltoluquinols as chemotaxonomic markers in the genus Tutour BI, Benslimane F, Gouleau MP, Gouygou JP, Saadan B,
Cystoseira: C. barbatula and C. barbata. Gazz. Chim. Ital. 120: Quemeneur F (1998) Antioxidant and pro-oxidant activities of
912. brown algae, Laminaria digitata, Himanthalia elongata, Fucus
Amico V, Piattelli M, Neri P, Ruberto G (1988b) Meroterpenoids vesiculosus, Fucus serratus and Ascophyllum nodosum. J. appl.
from Cystoseira spp. J. Nat. Prod. 51(1): 191192. Phycol. 10: 121129.
Amico V, Piattelli M, Neri P, Ruberto G, Mayol L (1986) Novel Yan X, Chuda Y, Suzuki M, Nagata T (1999) Fucoxanthin as the ma-
metabolites from the marine genus Cystoseira Application of jor antioxidant in Hijikia fusiformis, a common edible seaweed.
two-dimensional 1 H13 C correlation to the structure elucida- Biosci. Biotechnol. Biochem. 63: 605607.
tion. Tetrahedron 42(21): 60156020. Yen GC, Duh PD, Tsai CL (1993) Relationship between antioxidant
Anggardiredja J, Andyani R, Muaswanah H (1997) Antioxidant activity and maturity of peanut hulls. J. agric. Food Chem. 41:
activity of Sargassum polycystum and Laurencia obtusa from 6770.
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.