Food

Chemistry
Food Chemistry 98 (2006) 545–550
www.elsevier.com/locate/foodchem

Antioxidant properties of dried product of Ôhaba-noriÕ, an

edible brown alga, Petalonia binghamiae (J. Agaradh) Vinogradova
Takashi Kuda *, Tomoko Hishi, Sayuri Maekawa
Department of Food Science, Ishikawa Prefectural University, Suematsu 1-308, Nonoichi, Ishikawa 921-8836, Japan

Received 14 February 2005; received in revised form 15 June 2005; accepted 15 June 2005

Abstract

Dried Ôhaba-noriÕ Petalonia binghamiae, a brown alga, is a traditional food in the fisheries towns in Japan. To determine the
antioxidant properties of the dried P. binghamiae, assays for antioxidant activities, including ferrous-reducing power, ferrous ion
chelating, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging and scavenging of a superoxide anion radical-generated by
non-enzymatic system were tested in this study. A water extract solution contained total phenols at about 75 lmol phloroglucinol
equivalents/g dry sample and showed strong antioxidant activities in the reducing power, DPPH radical and superoxide anion
radical scavenging assays. The antioxidant activities were detected in high-molecular (>100 kDa), 10–30 kDa, and low-molecular
(<5 kDa) fractions and were correlated with, not only phenolic compounds, but also brown compounds. The radical- scavenging
activities were increased by heat treatment at 121 C for 1 h. These results suggest that P. binghamiae is both a useful seafood
and a healthy food with antioxidant activity.
 2005 Elsevier Ltd. All rights reserved.

Keywords: Petalonia binghamiae; Antioxidant property; Radical scavenging activity; Reducing power

1. Introduction Oxidative modification of DNA, proteins, lipid and

small cellular molecules by reactive oxygen species
A brown alga, Petalonia binghamiae (J. Agaradh) (ROS) plays a role in a wide range of common diseases
Vinogradova called Ôhaba-noriÕ in Japan, is widely dis- and age-related degenerative conditions (Borek, 1993).
tributed in the Pacific, for example along the coasts of These include cardiovascular disease, inflammatory con-
Japan, China and West of the USA (Segawa, 1996). ditions, and neurodegenerative diseases, such as Alzhei-
The shape of P. binghamiae is an aggregate of several merÕs disease (Richardson, 1993), mutations and cancer
leaves that are 15–50 mm in width and 100–250 mm in (Byress & Guerrero, 1995). Though there are publica-
length. Although P. binghamiae grows well along many tions about the antioxidant activity of seaweeds (Yan,
coasts of Japan and other countries, it is consumed as an Nagata, & Fan, 1998), there are few reports about anti-
edible alga and traditional food only in the fisheries oxidant activities in dried algal products (Kuda,
town areas. Usually, the alga is eaten after drying and Tsunekawa, Hishi, & Araki, 2005), and these studies
roasting lightly, like a dried product of ÔnoriÕ Porphyra are mainly confined to non-edible and/or fresh raw sea-
spp., a red alga, that is one of the major algal products weeds. It is reported that drying and storage decrease
(Kitamura, Myouga, & Kamei, 2002). the antioxidant compounds and activities (Araki, 1983).
There are some reports about fucoxanthin-related
*
Corresponding author. Tel.: +81 76 227 7220; fax: +81 76 227
compounds (Mori et al., 2004), including retinoic acid
7410. (vitamin A) in P. binghamiae. These compounds have
E-mail address: [email protected] (T. Kuda). inhibitory activities against mammalian replicative

0308-8146/$ - see front matter  2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2005.06.023
546 T. Kuda et al. / Food Chemistry 98 (2006) 545–550

DNA polymerases (Murakami et al., 2002). However, 2.4. Determination of the amount of total phenolic
there are hardly any reports about any functional activ- compounds, brown compounds, protein, saccharides and
ity, such as antioxidant activity, in dried products of P. minerals
binghamiae.
The aim of the present work was to evaluate the prof- Total phenolic compound concentrations were deter-
itable properties of P. binghamiae for human food. We mined as described previously (Kuda et al., 2005; Oki
investigated the antioxidant activities of water extract et al., 2002). Briefly, 0.4 ml of 10% Folin–Chiocalteu
and ethanol extract by ferrous-reducing power assay, solution was added to 0.2 ml of a sample solution. After
ferrous ion chelating assay, DPPH radical assay, and an interval of 3 min, 0.8 ml of a 10% sodium carbonate
superoxide anion-scavenging assay. These antioxidant was added. The mixture was allowed to stand for 30 min
assays employ methodology widely used for plants and at ambient temperature and the absorbance was then
processed foods. Effects of heating on the antioxidant measured at 750 nm. The phenolic content was ex-
activities were also examined. pressed as PG equivalent (Eq). To determine the relative
content of brown compounds in P. binghamiae, 0.2 ml of
WE was put into a microplate well and then absorbance
2. Materials and methods was measured at 490 nm with reference to absorbance at
655 nm.
2.1. Material Protein content in the individual fractions was mea-
sured with a DC protein assay kit (Bio-Rad, CA). Total
P. binghamiae was harvested in Wajima city (located saccharide was determined by the phenol–sulfuric meth-
in the temperate zone and facing the Sea of Japan), od (Dubois, Gilles, Hamilton, Rebens, & Smith, 1956).
Ishikawa, Japan in April, 2004. The harvested material Salinity was measured by a salt meter (ES-421, Atago,
was spread in a mesh bottom frame about 25 · 25 cm Tokyo). Concentrations of sodium and potassium were
and dried like ÕnoriÕ. The dried product was purchased determined by ion meters (C-122 and C-131, Horiba,
from a retail shop in Wajima and used in this study. Kyoto). Calcium and magnesium were measured with
commercially available kits (for Ca and Mg Test-Wako
2.2. Chemicals series, Wako Pure Chemical, Osaka).

(+)-Catechin (CA), Folin–CiocalteuÕs phenol reagent, 2.5. Reducing power

the stable radical DPPH, nitroblue tetrazolium salt
(NBT), phenazine methosulphate (PMS), 3-(2-pyridyl)- Total reducing power was determined as described by
5,6-di(p-sulfophenyl)-1,2,4-triazine, disodium salt (ferro- Zhu, Hackman, Ensunsa, Holt, and Keen (2002), but
zine) and ethylendiaminetetraacetic acid (EDTA) were modified slightly. Briefly, each 0.2 ml of the sample solu-
purchased from Sigma–Aldrich Co. (St. Louis, MO). tion was mixed with 0.2 ml of phosphate buffer (0.2 M,
Phloroglucinol dihydrate (PG) and Potassium ferricya- pH 7.2) and 0.2 ml of 1% potassium ferricyanide. The
nide were purchased from Wako Chemicals Co., Ltd. mixture was then incubated at 50 C for 20 min. After-
(Osaka, Japan). Other reagents were of analytical grade. wards, 0.2 ml of 10% trichloroacetic acid was added to
mixture. Finally, 0.125 ml of the mixture and 0.125 ml
2.3. Preparation of sample extract distilled water were put into a 96-well microplate and
0.02 ml of 0.1% FeCl3 was added. Increased absorbance
The dried product sample (2 g) was weighed and at 655 nm of the reaction mixture indicated increased
50 ml of distilled water or ethanol were added. The reducing power. PG was used as positive control.
water extract (WE) and ethanol extract (EE) solutions
were collected after shaking for 1 h at room temperature 2.6. Ferrous ion chelating activity
and centrifugation (2220 · 10 min). The sample prepara-
tion was replicated three times. The WE and EE solu- The method of Decker and Welch (1990) was used. To
tions were dark brown and dark green, respectively. a sample solution (0.1 ml), distilled water (0.1 ml) and
A portion (6 ml) of the WE solution was fractionated 0.5 mM FeCl2 (0.025 ml) were added. After measurement
into six fractions (each 6 ml) of molecular weights, that of absorbance at 550 nm, 2.5 mM ferrozine was added.
were >100, 50–100, 30–50, 10–30, 5–10 kDa, and After 20 min at room temperature, the absorbance was
<5 kDa, using an ultrafiltration system, VIVASPIN 6 measured. EDTA was used as positive control.
(Sartorius AG, Goettingen, Germany).
To determine the effect of heat treatment on the anti- 2.7. DPPH radical-scavenging activity
oxidant activities, the WE and EE were extracted at
85 C and 121 C (WE85, WE121, EE85 and EE121, DPPH radical-scavenging activity was determined by
respectively). the method of Blois (1958) with slight modification.
 T. Kuda et al. / Food Chemistry 98 (2006) 545–550 547

Thirty minutes after adding 1 mM DPPH/methanol Table 2

solution (0.025 ml) to the sample solutions (0.2 ml), Antioxidant properties of dried Ôhaba-noriÕ P. binghamiae
absorbance was measured at 550 nm. If the mixture WE EE
was turbid, the absorbance was measured after Ferrous-reducing power 94.2 ± 2.9 18.9 ± 0.1
centrifugation. (lmol PG Eq/g dry sample)
Ferrous ion chelating activity 2.32 ± 0.33 <2
(lmol EDTA Eq/g dry sample)
2.8. Superoxide anion radical-scavenging activity DPPH radical-scavenging 2.73 ± 0.49 0.53 ± 0.16
(mmol PG Eq/g dry sample)
The non-enzymatic generation of superoxide anion Superoxide anion scavenging 80.0 ± 1.9 5.9 ± 1.7
was measured by the method of Robak and Gryglewski (lmol CA Eq/g dry sample)
(1988). Sample solution (0.1 ml) was treated with 0.1 ml WE, water extract solution; EE, ethanol extract solution.
of 0.1 M phosphate buffer (pH 7.2), 0.025 ml of 2 mM PG, phloroglucinol; Eq, equivalent; CA, (+) catechin.
NADH and 0.025 ml of 0.5 mM NBT, and absorbance Values are means and SD (n = 3).
at 550 nm was measured as a blank value. After a
3 min incubation with 0.025 ml of 0.03 mM PMS, the DPPH has been used extensively as a radical to eval-
absorbance was again measured. uate reducing substances (Cotelle et al. (1996)). In most
cases, irrespective of the stage in the oxidative chain in
which the antioxidant action is assessed, most non-enzy-
3. Results and discussion matic antioxidative activity, such as scavenging free rad-
icals or inhibition of peroxidation, is mediated by redox
3.1. Chemical compounds in extract solutions reactions (Zhu et al. (2002)). In most organisms, super-
oxide anion radical is converted to hydrogen peroxide
The total phenolic contents in WE and EE were 73.5 by superoxide dismutase. In the absence of transition
and 21.8 lmol PG Eq/g dry sample, respectively (Table metal ions, hydrogen peroxide is fairly stable. However,
1). The other main compounds in WE were saccharides hydroxyl radicals can be formed by the reaction of
(polysaccharides). Protein and mineral contents in WE superoxide with hydrogen peroxide in the presence of
were not so high. Potassium content was four times metal ions, usually ferrous or copper (Macdonald, Gal-
higher than sodium content. ley, & Webster (2003)).
Ferrous ion chelating activity of both WE and EE
3.2. Antioxidant properties of the water and ethanol was not so high comparing with Ôkayamo-noriÕ Scytosi-
extract solutions phon lomentaria (Kuda et al. (2005)). The metal binding
capacities of dietary fibers are well known and the inhib-
Ferrous-reducing power, DPPH radical scavenging itory effects on ferrous absorption of algal dietary fibers
activity and superoxide anion radical scavenging activity have been reported (Harmuth-Hoene & Schelenz
of WE were higher than that of EE (Table 2). The phe- (1980)). It is considered that the amount and/or ferrous
nolic content, ferrous-reducing power and radical scav- binding capacities of water-soluble polysaccharides,
enging activity in WE was higher than that of such as alginate, fucoidan and laminaran, in P. bingh-
common brown algae, such as Fucus, Laminaria, Unda- amiae were lower than ones of S. lomentaria.
ria, (Jiménez-Escrig, Jiménez-Jiménez, Pulido, & Saura-
Calixto (2001)), besides of ÔkajimeÕ Ecklonia cava (Nagai 3.3. Antioxidant properties of individual P. binghamiae
& Yukimoto (2003)). molecular weight fractions

Table 1 As summarized in Table 1, WE of P. binghamiae was

Chemical compounds in extractions of dried Ôhaba-noriÕ P. binghamiae rich in phenolic compounds. With respect to the six
WE EE molecular weight fractions isolated, the greatest amount
of phenolic compounds was found in the >5 kDa, and
Total phenolic compounds 75.3 ± 7.5 21.8 ± 0.3
(lmol PG Eq/g dry sample) >100 kDa fractions (Fig. 1(a)). The lowest amount of
Soluble saccharide (mg/g dry sample) 78.1 ± 15.2 – phenolic compounds was measured in the 50–100- and
Soluble protein (mg/g dry sample) 5.15 ± 0.37 – 30–50-kDa fractions.
Salinity (mg/g dry sample) 53 ± 1 – Absorbance at 490 nm referenced by the absorbance at
Na 3.3 ± 0.1 –
655 nm was used as brown compounds (Fig. 1(b)). About
K 11.3 ± 0.3 –
Mg 1.68 ± 0.05 – 60%, 20% and 10% of the brown compounds were in the
Ca 1.52 ± 0.19 – >100-, 10–30- and <5-kDa fraction, respectively.
WE, water extract solution; EE, ethanol extract solution. As shown in Fig. 1(c), the highest ferrous-reducing
PG, phloroglucinol; Eq, equivalent. power was observed in the >100 kDa, followed by <5,
Values are means and SD (n = 3). 10–30 and 5–10-kDa fractions. This result is thought
548 T. Kuda et al. / Food Chemistry 98 (2006) 545–550

Fig. 1. Antioxidant properties of individual molecular weight fractions from dried P. binghamiae. (a) Amount of total phenolic compounds. (b)
Relative content of brown compounds. (c) Ferrous-reducing power. (d) Ferrous ion chelating activity. (e) DPPH radical scavenging activity. (f)
Scavenging activity in non-enzymatic assay. Sample solution volumes in the assays c–f were 0.1, 0.1, 0.05 and 0.05 ml, respectively. Values are means
and SD (n = 3).

to correlate with the content of brown compounds The highest superoxide anion radical scavenging
rather than with the content of phenolic compounds, activity was shown in the >100-kDa fraction followed
though many researchers have suggested that there by the 10–30- and <5-kDa fractions (Fig. 1(f)). This re-
may be a relationship between the amount of total phe- sult is similar to that of the ferrous-reducing power
nolic compound and reducing power (Jiménez-Escrig (Fig. 1(c)).
et al., 2001; Zue et al., 2002).
The highest ferrous ion chelating activity was ob- 3.4. Effect of extraction temperature on the antioxidant
served in the 10–30-kDa fraction, followed by <5-kDa properties
fraction (Fig. 1(d)). Interestingly, the chelating activity
of the 10–30-kDa fraction (95%) was higher than that To determine the effect of heating on the antioxida-
of the WE (52%) at the same sample solution volume tive activity of P. binghamiae, WE and EE were obtained
(0.1 ml) in this assay. from dried P. binghamiae at room temperature, 85 and
The highest DPPH scavenging activity was shown in 121 C for 1 h.
the <5-kDa fraction, followed by <100- and 10–30-kDa The total phenolic compounds in WE extracted at
fractions (Fig. 1(e)). This result is thought to correlate 121 C (WE121) was 65% higher than that of WE ex-
with the content of phenolic compounds rather than tracted at room temperature (WERT) (Fig. 2(a)). On
with the content of brown compounds. As with reducing the other hand, the content in WE heated at 85 C
power, it has been reported that the amount of DPPH- (WE85) was decreased slightly. The phenolic content
scavenging activity is dependent on the phenolic concen- in EE was not so affected by the extraction tempera-
tration of the algal samples (Jiménez-Escrig et al., 2001). ture. The brown compound was increased about 2.5
 T. Kuda et al. / Food Chemistry 98 (2006) 545–550 549

Fig. 2. Effect of heating on the antioxidant activities of water extract (open square) and ethanol extract (semi-closed square) solutions from dried P.
binghamiae. (a) Amount of total phenolic compounds. (b) Relative content of brown compounds. (c) Ferrous-reducing power. (d) Ferrous ion
chelating activity. (e) DPPH radical scavenging activity. (f) Scavenging activity in non-enzymatic assay. Sample solution volumes in the assays c–f
were 25, 50, 1.25 and 12.5 ll, respectively. R.T., room temperature. Values are means and SD (n = 3).

times in WE121 (Fig. 2(b)). It is considered that the DPPH radical scavenging activities in WE were 20%
brown compound was generated by the Maillard and 50% increased by heating at 85 and 121 C, respec-
reaction. tively (Fig. 2(e)). On the other hand, the activity in EE
As shown in Fig. 2(c), the reducing power was not in- was decreased slightly by the heat treatment. Superoxide
creased in WE121. Furthermore, the reducing power de- anion radical-scavenging activity in WE was also clearly
creased by 35% in WE85. This result may be related to increased by the heating (Fig. 2(f)).
phenolic compound content. Jiménez-Escrig et al. It is reported that drying and storage decrease antiox-
(2001) reported that the phenolic content and reducing idant compounds, such as ascorbic acid and fucoxan-
power in Fucus were decreased by drying at 50 C for tins, and their activities (Araki, 1983). However, there
48 h, and storage at room temperature. are many reports about radical-scavenging activity of
The chelating activities were about 65% and 100% in- brown compounds (pigments) induced by Maillard reac-
creased in WE85 and WE121, respectively (Fig. 2(d)). tion (Jing & Kitts, 2002; Morales & Babbel, 2002). It is
Perhaps, the increased activity was brought about by considered that the brown compounds having radical
increasing solubility of polysaccharides. We had re- scavenging activity are generated by the Maillard reac-
ported that the crude alginate and fucoidan from Scyto- tion in WE during the heating.
siphon lomentaria, heated at 121 C for 15 min, have the Although there are several reports about antioxidant
ferrous ion binding activity (Kuda et al., 2005). activity of raw edible brown algae, such as ÔkombuÕ
550 T. Kuda et al. / Food Chemistry 98 (2006) 545–550

Laminaria, ÔwakameÕ Undaria and ÔkajimeÕ Ecklonia Jiménez-Escrig, A., Jiménez-Jiménez, I., Pulido, R., & Saura-Calixto,
(Jiménez-Escrig et al., 2001; Kang et al., 2004; Yan F. (2001). Antioxidant activity of fresh and processed edible
seaweeds. Journal of the Science of Food and Agriculture, 81,
et al., 1998), we believe that the algal foods circulating 530–534.
as dried product should be examined after the drying pro- Jing, H., & Kitts, D. D. (2002). Chemical and biological properties of
cess. Usually, these brown algae are dried and eaten after casein-sugar Maillard reaction product. Food and Chemical Tox-
swelling with 20–40 and more volume of water. On the icology, 40, 1007–1015.
other hand, P. binghamiae is eaten after drying and only Kang, H. S., Chung, H. Y., Kim, J. Y., Son, B. W., Jung, H. A., &
Choi, J. S. (2004). Inhibitory phlorotannins from the edible brown
light roasting. alga Ecklonia stolonifera on total reactive oxygen species (ROS)
In summary, our observations demonstrate that WE generation. Archives of Pharmacal Research, 27, 194–198.
and its >100- <5- and also 10–30-kDa fractions have Kitamura, E., Myouga, H., & Kamei, Y. (2002). Polysaccharolytic
reducing power and radical scavenging activity. Further- activities of bacterial enzymes that degrade the cell walls of
more, the radical scavenging and ferrous-chelating activ- Phthium porphyrae, a causative fungus of red rot disease in
Porphyra yezoensis. Fisheries Science, 68, 436–445.
ities are promoted by heat treatment such as retortring. Kuda, T., Tsunekawa, M., Hishi, T., & Araki, Y. (2005). Antioxidant
In general, it is considered that the antioxidant activities properties of dried Ôkayamo-noriÕ, a brown alga Scytosiphon
depend on the phenolic and/or brown compounds. lomentaria (Scytosiphonales, Paheophyceae). Food Chemistry, 59,
However, the antioxidant activities of sulfated polysac- 617–622.
charides, such as fucoidan have been reported (Kuda Macdonald, J., Galley, H. F., & Webster, N. R. (2003). Oxidative
stress and gene expression in sepsis. British Journal of Anaesthesia,
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that P. binghamiae is a useful healthy sea vegetable hav- Maillard reaction mixture in a hydrophilic media. Journal of
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H., Kawasaki, M., et al. (2002). Vitamin A-related compounds,
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