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Antioxidant potential of solvent extracts of Kappaphycus
alvarezii (Doty) Doty – An edible seaweed
K. Suresh Kumar, K. Ganesan, P.V. Subba Rao
*
Marine Biotechnology and Ecology Discipline, Central Salt and Marine Chemicals Research Institute (CSIR), Bhavnagar – 364 002, Gujarat, India
Received 11 May 2007; received in revised form 30 June 2007; accepted 6 August 2007
Abstract
Various solvent extracts of Kappaphycus alvarezii, an edible red seaweed (family Solieriaceae) were screened for total phenol content
and antioxidant activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferrous ion chelating activity, reducing power and antioxidant
activity assays in a linoleic acid system with ferrothiocyanate reagent (FTC). The total phenol content of different extracts of K. alvarezii
varied from 0.683 ± 0.040% to 2.05 ± 0.038%. The radical-scavenging activity of ethanol extract was, as IC
50
3.03 mg ml
1
, whereas that
of the water extract was IC
50
4.76 mg ml
1
. Good chelating activity was recorded for methanol extract (IC
50
3.08 mg ml
1
) wherein
67.0 ± 0.924% chelation was obtained using 5.0 mg ml
1
of extract. The reducing power of the samples was in the following order:
BHT > methanol > ethanol > ethyl acetate > water > hexane. But, in the linoleic acid system, the ethanol extract proved superior to
the synthetic antioxidants butylated hydroxytoluene (BHT). Hence, these extracts could be considered as natural antioxidants and
may be useful for curing diseases arising from oxidative deterioration.
Ó2007 Elsevier Ltd. All rights reserved.
Keywords: Kappaphycus alvarezii; Antioxidant activity; Total phenolics; Free radicals; Reducing power
1. Introduction
All living organisms contain complex systems of antiox-
idant enzymes. Some of these systems, e.g. the thioredoxin
system, are conserved throughout evolution and are
required for life. Antioxidants in biological systems have
multiple functions, including defending against oxidative
damage and participating in the major signalling pathways
of cells. One major action of antioxidants in cells is to pre-
vent damage caused by the action of reactive oxygen spe-
cies. Reactive oxygen species include hydrogen peroxide
(H
2
O
2
), the superoxide anion (O
2), and free radicals, such
as the hydroxyl radical (
OH). These molecules are unstable
and highly reactive, and can damage cells by chain reac-
tions, such as lipid peroxidation, or formation of DNA
adducts that could cause cancer-promoting mutations or
cell death. In order to reduce or prevent this damage, all
cells invariably contain antioxidants.
Lipid oxidation by reactive oxygen species (ROS), such
as superoxide anion, hydroxyl radicals, and hydrogen per-
oxide, causes a decrease in nutritional value of lipids, in
their safety and appearance. In addition, it is the predom-
inant cause of qualitative decay of foods, which leads to
rancidity, toxicity, and destruction of biochemical compo-
nents important in physiologic metabolism. Free radical-
mediated modification of DNA, proteins, lipids and small
cellular molecules are associated with a number of patho-
logical processes, including atherosclerosis, arthritis, diabe-
tes, cataractogenesis, muscular dystrophy, pulmonary
dysfunction, inflammatory disorders, ischemiareperfusion
tissue damage, and neurological disorders, such as Alzhei-
mer’s disease (Frlich & Riederer, 1995).
Antioxidants are classified by the products they form
on oxidation (these can be antioxidants themselves, inert,
0308-8146/$ - see front matter Ó2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2007.08.016
*
Corresponding author.
E-mail address: pvsubbarao@csmcri.org (P.V.S. Rao).
www.elsevier.com/locate/foodchem
Available online at www.sciencedirect.com
Food Chemistry 107 (2008) 289–295
Food
Chemistry
or pro-oxidant), by what happens to the oxidation prod-
ucts (the antioxidant may be regenerated by different
antioxidants or, in the case of ‘‘sacrificial”antioxidants,
its oxidized form may be broken down by the organism)
and how effective the antioxidant is against specific free
radicals. Several synthetic antioxidants, such as butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT),
and tert-butylhydroquinone (TBHQ), are commercially
available and currently used. However, these antioxidants
have been restricted for use in foods as they are sus-
pected to be carcinogenic. Some toxicological studies
have also implicated the use of these synthetic antioxi-
dants in promoting the development of cancerous cells
in rats. These findings, together with consumers’ interests
in natural food additives, have reinforced the efforts for
the development of alternative antioxidants of natural
origin (Huang & Wang, 2004). An immense number of
marine flora and fauna are reported to have a wide spec-
trum of interesting biological properties. In folk medi-
cine, seaweeds have been used for a variety of remedial
purposes, e.g. for the treatment of eczema, gallstone,
gout, crofula, cooling agent for fever, menstrual trouble,
renal problems and scabies (Chapman & Chapman,
1976).
Seaweeds are rich in polysaccharides, minerals, proteins
and vitamins. Documented antioxidant activity would ele-
vate their value in the human diet as food and pharmaceu-
tical supplements (Yan, Nagata, & Fan, 1998). Few reports
are available on the antioxidant potential of seaweeds
(Jimenez-Escrig, Jimenez-Jimenez, Pulido, & Saura-Cali-
xto, 2001). Ismail and Hong (2002) reported antioxidant
activity of four commercial edible seaweeds, namely Nori
(Porphyra sp.), Kumbu (Laminaria sp.), Wakame (Undaria
sp.) and Hijiki (Hijikia sp.).
The Rhodophyta (red algae) are a distinct eukaryotic
lineage, characterized by the accessory photosynthetic pig-
ments phycoerythrin, phycocyanin and allophycocyanins
arranged in phycobilisomes. They contain a large assem-
blage of species that predominate in the coastal and con-
tinental shelf areas of tropical, temperate and cold-water
regions. Red algae are ecologically significant as primary
producers, providers of structural habitat for other mar-
ine organisms, and they play an important role in the pri-
mary establishment and maintenance of coral reefs. Some
red algae are economically important as providers of food
and gels (Wilson, 2000). For this reason, extensive farm-
ing and natural harvest of red algae occur in numerous
areas of the world. Kappaphycus alvarezii, an economi-
cally important red tropical seaweed, which is highly
demanded for its cell wall polysaccharide, is the most
important source of kappa carrageenan. The world
production of Kappaphycus species is approximately
28000 tons per annum. This seaweed accounts for the
largest consumption worldwide (McHugh, 1987). It is eas-
ily accessible, in huge amounts, for food and pharmaceu-
tical applications. The present study deals with
antioxidant properties of K. alvarezii.
2. Materials and methods
2.1. Collection of samples
K. alvarezii was collected from a cultivation site at Port
Okha (L 22°28.5280N; L 069°04.3220E) located on the north
west coast of India during April, 2006. The sample was
thoroughly washed with seawater to remove epiphytes
and dirt particles, followed by shade-drying for two days.
It was then brought to the laboratory, oven-dried at 70 °C
for 4 h to obtain a constant weight and pulverized in the
grinder (size 2 mm). This sample was used for determina-
tion of phenolic content, as well as for antioxidant studies.
The chemicals used in these studies were of analytical grade.
2.2. Preparation of extracts
The pulverized moisture-free sample (20 g) was
extracted with 200 ml of individual solvents using a Soxhlet
extractor. The extraction was repeated many times to
obtain a sizable quantity of extract. Consequently, the
extract was concentrated in a rotary evaporator at 40 °C.
Different solvents were used for the preparation of extracts
to determine the antioxidant efficacy of K. alvarezii. All the
experiments were conducted in triplicate.
2.3. Determination of total phenol
Total phenolic content was estimated by Folin–Ciocal-
teau method (Singleton & Rossi, 1965). To 6.0 ml of
double-distilled water, 0.1 ml of sample and 0.5 ml of
Folin–Ciocalteau reagent were mixed, followed by the addi-
tion of 1.5 ml of Na
2
CO
3
(20 g 100 ml
1
water) and the
volume was made up to 10.0 ml with distilled water. After
incubation for 30 min at 25 °C, the absorbance was
measured at 760 nm and the total phenolic content was cal-
culated with a gallic acid standard and expressed as a per-
centage of total phenols obtained on a dry weight basis.
2.4. DPPH radical scavenging assay
DPPH
-scavenging potential of different fractions was
measured, based on the scavenging ability of stable 1,1-
diphenyl-2-picrylhydrazyl (DPPH) radicals by K. alvarezii
antioxidants. The ability of extracts to scavenge DPPH rad-
icals was determined by the method of Blois (1958). Briefly,
1 ml of 1 mM methanolic solution of DPPH
was mixed
with 1 ml of extract solution (containing 0.5–5.0 mg ml
1
of dried extract). The mixture was then vortexed vigorously
and left for 30 min at room temperature in the dark. The
absorbance was measured at 517 nm and activity was
expressed as percentage DPPH
-scavenging activity relative
to the control, using the following equation:
%Radical scavenging activity
¼AControl ASample=AControl
100
290 K.S. Kumar et al. / Food Chemistry 107 (2008) 289–295
2.5. Ferrous ion-chelating activity
Iron-chelating abilities of methanol, ethanol and ethyl
acetate extracts of K. alvarezii were used for the present
investigation. The chelating of ferrous ions by the extracts
and standards was estimated by the method of Dinis,
Madeira and Almeida (1994). Extracts were added to a
solution of 2 mM FeCl
2
(0.05 ml). The reaction was initi-
ated by the addition of 5 mM ferrozine (0.2 ml) and the
mixture was shaken vigorously and left standing at room
temperature for 10 min. After the mixture had reached
equilibrium, the absorbance of the solution was then mea-
sured at 562 nm. The percentage inhibition of ferrozine–
Fe
2+
complex formation was determined using the follow-
ing formula:
%Inhibition ¼1A1Sample=A0Control
100
where A
0
was the absorbance of the control and A
1
was the
absorbance in the presence of the sample extracts and stan-
dards. The control contained FeCl
2
and ferrozine, with
complex formation molecules.
2.6. Reducing power
Extracts of K. alvarezii were prepared using methanol,
ethanol, water, ethyl acetate and hexane. The reductive
potential of extracts was determined by the method of
Oyaizu (1986). The different concentrations of extracts
(0.5–25 mg ml
1
) were mixed with phosphate buffer
(2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide
[K
3
Fe(CN)
6
] (2.5 ml, 1%). The mixture was incubated at
50 °C for 20 min. A portion (2.5 ml) of trichloroacetic acid
(10%) was added to the mixture, which was then subjected
to centrifugation (10 min, 1000g). The upper layer of solu-
tion (2.5 ml) was mixed with distilled water (2.5 ml) and
FeCl
3
(0.5 ml, 0.1%), and the absorbance was measured
at 700 nm. Higher absorbance of the reaction mixture indi-
cated greater reductive potential.
2.7. Antioxidant activity in the linoleic acid system with
ferrothiocyanate reagent (FTC)
Ethanolic extract of K. alvarezii was subjected to the
assay adopted by Osawa and Namaki (1983). The extract
(4 mg) was dissolved in 99.5% ethanol and mixed with
2.5% linoleic acid in 99.5% ethanol (4.1 ml), 0.05 M phos-
phate buffer (pH 7, 8 ml) and distilled water (3.9 ml) and
kept in screw-cap containers under dark conditions at
40 °C; 0.1 ml of this solution was added to 9.7 ml of 75%
ethanol and 0.1 ml of 30% ammonium thiocyanate. After
3 min, 0.1 ml of 0.02 M ferrous chloride in 3.5% hydrochlo-
ric acid was added to the reaction mixture, the absorbance
of the red colour was measured at 500 nm in the spectro-
photometer every two days. The control and standard were
subjected to the same procedure except that for the control,
there was no addition of sample and, for the standard,
4 mg of sample was replaced with 4 mg of butylated
hydroxy toluene (BHT), used as a positive control. Absor-
bance was measured at intervals of 2 days. The percent
inhibition of linoleic acid peroxidation was calculated as:
Inhibition ð%Þ
¼100 ðabsorbance increase of the sample½
=absorbance increase of the controlÞ100
The IC
50
value represented the concentration of the com-
pounds that caused 50% inhibition. All experiments were
carried out in triplicate.
2.8. Statistical analysis
For the extract, three samples were prepared for each
experiment. The data were presented as mean ± standard
deviation.
3. Results and discussion
3.1. Antioxidant activity
The antioxidant activity is system-dependent. Moreover,
it depends on the method adopted and the lipid system
used as substrate (Singh, Maurya, de Lampasona, & Cata-
lan, 2006). Hence, the following different methods have
been adopted in order to assess the antioxidative potential
of K. alvarezii extracts.
3.2. Total phenol content
A number of studies have focussed on the biological
activities of phenolic compounds, which are potential anti-
oxidants and free radical-scavengers (Ka
¨hko
¨nen et al.,
1999; Rice-Evans, Miller, Bolwell, Bramley, & Pridham,
1995; Sugihara, Arakawa, Ohnishi, & Furuno, 1999). The
total phenol content was maximum when a mixture of
chloroform and methanol (2:1) was used (2.05 ± 0.038%)
followed by ethanol (1.94 ± 0.029%), methanol
(1.79 ± 0.77%), n-propanol (1.40 ± 0.040%) and ethyl ace-
tate (1.09 ± 0.597%). Extracts obtained using other sol-
vents, namely acetone, n-hexane and chloroform, showed
<1% total phenol content (Table 1).
3.3. Scavenging effect on 1,1-diphenyl-2-picrylhydrazyl
radical (DPPH
)
The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical is a
stable radical with a maximum absorbance at 517 nm that
can readily undergo reduction by an antioxidant. Because
of the ease and convenience of this reaction, it has now
widespread use in the free radical-scavenging activity
assessment (Brand-Williams, Cuvelier, & Benset, 1995).
The radical-scavenging activity of K. alvarezii extract is
shown in Fig. 1 and expressed as percentage reduction of
the initial DPPH
absorption by the tested compound.
The best radical-scavenging activity could be obtained in
K.S. Kumar et al. / Food Chemistry 107 (2008) 289–295 291
the ethanol extract (IC
50
3.03 mg ml
1
), followed by meth-
anol (IC
50
4.28 mg ml
1
). Extracts obtained using water
also showed equivalent scavenging activity (IC
50
4.76 mg ml
1
). These values were lower than those
obtained using BHT (IC
50
2.83 mg ml
1
), but the IC
50
val-
ues of the methanol and water extracts were comparable
with a-tocopherol (IC
50
4.55 mg ml
1
). The extracts of K.
alvarezii showed better radical-scavenging activity than
did the extract of Palmaria palmata (dulse) IC
50
–
12.5 mg ml
1
(Yuan, Carrington, & Walsh, 2005a), and
purified extract of Ecklonia cava IC
50
– 5.49 10
3
lgml
1
(c.f. Suja, Jayalekshmy, & Arumughan, 2005). Ragan and
Glombitza (1986) reported the radical-scavenging activity
of seaweeds to be mostly related to their phenolic contents.
On the other hand, Siriwardhana, Lee, Kim, Ha, and Jeon
(2003) and Lu and Foo (2000) reported a high correlation
between DPPH radical-scavenging activities and total poly-
phenolics r= (0.971). In the present study, the linear
regression analysis of DPPH
-scavenging (i.e EC
50
values)
with the total phenol content (gallic acid equivalents) gave
an rvalue of 0.937, showing statistically significant correla-
tion. K. alvarezii is the main industrial source of carra-
geenan (having alternating D-galactose 4-sulphate and
3,6-anhydro D-galactose residues), which may also contrib-
ute to the antioxidant potential of this seaweed. Compo-
nents, such as low molecular weight polysaccharides,
pigments, proteins or peptides, also influence the antioxi-
dant activity (Siriwardhana et al., 2003).
3.4. Metal ion-chelating activity
All the extracts demonstrated reasonable ferrous ion che-
lating efficacy (Fig. 2). The ascorbic acid extract demon-
strated best ferrous chelating efficacy (IC
50
2.88 mg ml
1
)
followed by methanol, ethanol and ethyl acetate (IC
50
3.08, 3.83 and 4.38 mg ml
1
, respectively). Iron is known
to generate free radicals through the Fenton & Haber-Weiss
reaction. Metal ion-chelating activity of an antioxidant
molecule prevents oxyradical generation and the conse-
quent oxidative damage. Metal ion-chelating capacity plays
a significant role in the antioxidant mechanism since it
reduces the concentration of the catalyzing transition metal
in LPO. It is reported that chelating agents that form r-
bonds with a metal, are effective as secondary antioxidants
since they reduce the redox potential, thereby stabilizing the
oxidized form of the metal ion (Srivastava, Harish, & Shiva-
nandappa, 2006). Metal-binding capacities of dietary fibres
are well known, e.g. the inhibitory effects on ferrous absorp-
tion of algal dietary fibres such as carrageenan, agar and
alginate, were reported (Harmuth-Hoene & Schelenz,
1980). In this present study, the carrageenan might have
caused the decrease of ferrous ion in the assay system.
3.5. Measurement of reducing potential
The reducing power of K. alvarezii extracts was concen-
tration-dependent (Fig. 3). As the concentration increased
from 0.5 to 5.0 mg ml
1
, there was an increase in absor-
bance with all the solvents except hexane. However, the
reducing powers of the samples were found to be in the fol-
lowing order: BHT (0.23–0.879) > methanol (0.07–
Table 1
Percent phenol content of K. alvarezii in various solvents
Solvents Total phenol (%)
Acetone 0.963 ± 0.058
n-Propanol 1.40 ± 0.040
Ethyl acetate 1.09 ± 0.597
n-Hexane 0.83 ± 0.048
Chloroform 0.683 ± 0.040
Methanol 1.79 ± 0.77
Ethanol 1.94 ± 0.029
Chloroform:methanol (2:1) 2.05 ± 0.038
Values are means of three replicate determinations; SD, standard
deviation.
Fig. 1. Antioxidant activities of different solvent extracts of K. alvarezii determined as DPPH radical-scavenging activity.
292 K.S. Kumar et al. / Food Chemistry 107 (2008) 289–295
0.74) > ethanol (0.333–0.44) > ethyl acetate (0.013–
0.467) > water (0.017–0.193) > hexane (0.017–0.16). It is
believed that antioxidant activity and reducing power are
related. Reductones inhibit LPO by donating a hydrogen
atom and thereby terminating the free radical chain reac-
tion (Srivastava et al., 2006).
3.6. Antioxidant activity in a linoleic acid system with
ferrothiocyanate reagent (FTC)
Peroxyl radicals are formed by a direct reaction of oxy-
gen with alkyl radicals. Decomposition of alkyl peroxides
also results in peroxyl radicals. Peroxyl radicals are good
oxidizing agents, having more than 1000 mV of standard
reduction potential (Decker, 1998). They can abstract
hydrogen from other molecules with lower standard reduc-
tion potentials. This reaction is frequently observed in the
propagation stage of lipid peroxidation. Cell membranes
are phospholipid bilayers with extrinsic proteins and are
the direct target of lipid oxidation (Girotti, 1998). As lipid
oxidation of cell membranes increases, the polarity of lipid
phase surface charge and formation of protein oligomers
increase; and molecular mobility of lipids, number of SH
groups, and resistance to thermal denaturation decrease.
Malonaldehyde, one of the lipid oxidation products, can
react with the free amino group of proteins, phospholipid,
and nucleic acids, leading to structural modifications,
which induce dysfunction of immune systems. The antiox-
idant effects of K. alvarezii extract and BHT on the perox-
idation of linoleic acid were investigated and the results are
presented in Fig. 4. The absorbance ranges recorded for
control, BHT and sample were 0.0087–0.0151, 0.0021–
0.0093 and 0.0037–0.0104, respectively. The ethanolic
extract of K. alvarezii showed higher inhibitory effect than
Fig. 2. Ferrous ion-chelating activities of different solvent extracts of K. alvarezii.
Fig. 3. Reducing powers of K. alvarezii extracts, along with a synthetic antioxidant.
K.S. Kumar et al. / Food Chemistry 107 (2008) 289–295 293
did the positive control, BHT. This might be due to the
presence of ascorbic acid and vitamin A (b-carotene) con-
tent in the extract of K. alvarezi (Fayaz et al., 2005).
Algal polysaccharides play an important role as free rad-
ical-scavengers in vitro and antioxidants for the prevention
of oxidative damage in living organisms. Their activity
depends on several structural parameters, such as the degree
of sulfation (DS), the molecular weight, the sulfation posi-
tion, type of sugar and glycosidic branching. Moreover,
some reports reveal that the sulfate and phosphate groups
in the polysaccharides lead to differences in their biological
activities. In vitro antioxidant activity of j-carrageenan oli-
gosaccharides and their oversulfated, acetylated, and phos-
phorylated derivatives have been reported by Yuan et al.
(2005b). They also reported that phosphorylated and sul-
fated glucans exhibited better antioxidant ability than did
glucans or other neutral polysaccharides, which indicates
that polyelectrolytes, such as glucan sulfate or phosphate,
might have increased scavenging activity. Moreover, the sul-
fate content of polysaccharides from Porphyra yezoensis
was reported to contribute to the antioxidant activity. The
cell wall of K. alvarezii is known to be constituted of carra-
geenan, a sulfated polysaccharide, which may contribute to
its antioxidant potential in addition to the presence of ascor-
bic acid, vitamin A and various phenolics.
4. Conclusion
In the present investigation, the various solvent extracts
of K. alvarezii exhibited excellent scavenging effect (%) by
DPPH
assay, reducing power, ferrous ion-chelating activ-
ity and antioxidant property in the linoleic acid system.
Thus they could be used in nutraceutical and functional
food applications. Since this is a preliminary study, a
detailed investigation on the compositions of each compo-
nent involved is absolutely necessary to establish appropri-
ate applications which may open new frontiers for human
consumption of this seaweed world-wide.
Acknowledgements
The authors are grateful to Dr. Pushpito Ghosh, Direc-
tor, Central Salt & Marine Chemicals Research Institute,
Bhavnagar, Gujarat, India for his constant support and
encouragement. They also appreciate Discipline Co-ordi-
nator, Marine Biotechnology and Ecology Discipline, for
providing research facilities and profusely thank the
Department of Biotechnology (Sanction No: BT/PR
3309/PID/03/139/2002), New Delhi, India for providing
financial assistance.
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