Antioxidant Activities and Phenolics Content of Eight Species

Antioxidant activities and phenolics content of eight species

of seaweeds from north Borneo

Patricia Matanjun & Suhaila Mohamed &

Noordin Mohamed Mustapha & Kharidah Muhammad &

Cheng Hwee Ming

Received: 1 March 2007 /Revised and Accepted: 10 August 2007 / Published online: 2 May 2008

# Springer Science + Business Media B.V. 2007

Abstract The antioxidant activity of eight edible species of

Malaysian North Borneo seaweeds obtained from Sabah

waters (Kudat, Tanjung Aru and Semporna) consisting of

three red seaweeds (Eucheuma cottonii, E. spinosum and

Halymenia durvillaei), two green seaweeds (Caulerpa

lentillifera and C. racemosa) and three brown seaweeds

(Dictyota dichotoma, Sargassum polycystum and Padina

sp.) were determined. Methanol and diethyl ether were used

as extraction solvent. The antioxidant activities were

determined by two methods, TEAC (trolox equivalent

antioxidant capacity) and FRAP (ferric reducing antioxidant power) assays. The total phenolic content of the

extract was determined according to the Folin–Ciocalteu

method and results were expressed as phloroglucinol

equivalents. The methanolic extracts of green seaweeds,

C. lentillifera and C. racemosa, and the brown seaweed, S.

polycystum showed better radical-scavenging and reducing

power ability, and higher phenolic content than the other

seaweeds. The TEAC and FRAP assays showed positive

and significantly high correlation (R

2

=0.89). There was a

strong correlation (R

2

=0.96) between the reducing power

and the total phenolic content of the seaweeds methanolic

dry extracts. These seaweeds could be potential rich sources

of natural antioxidants.

Keywords Seaweeds

. Antioxidant activity . TEAC . FRAP.

Phenolics

. North Borneo waters

Introduction

Reactive oxygen species (ROS) and oxidative stress have

been associated with the onset of a variety of chronic

disease states in humans, including coronary heart disease

(CHD), certain cancers, rheumatoid arthritis, diabetes,

retinopathy of prematurity, chronic inflammatory disease

of the gastrointestinal tract, as well as diseases associated

with cartilage, Alzeimer’s disease (Chauhan and Chauhan

2006), other neurological disorders and the ageing process

(Temple 2000). ROS are toxic as they can oxidize

biomolecules leading to cell death and tissue injury

(Morrisey and O’Brien 1998). Conversely, antioxidants

are believed to be protective because they may help to

protect the human body against damage by ROS (Halliwell

et al. 1995). Antioxidants from natural sources are preferred

by consumers (Kranl et al. 2005) due to concerns on the

toxic and carcinogenic effects of synthetic antioxidants (Ito

et al. 1986; Safer and Al-Nughamish 1999).

J Appl Phycol (2008) 20:367–373

DOI 10.1007/s10811-007-9264-6

P. Matanjun

School of Food Science and Nutrition, Universiti Malaysia Sabah,

Locked Bag 2073,

88999 Kota Kinabalu, Sabah, Malaysia

S. Mohamed (*)

: K. Muhammad

Faculty of Food Science & Technology,

Universiti Putra Malaysia,

UPM 43400 Serdang,

Selangor, Malaysia

e-mail: [email protected]

N. M. Mustapha

Faculty of Veterinary Medicine, Universiti Putra Malaysia,

UPM 43400 Serdang,

Selangor, Malaysia

C. H. Ming

Department of Physiology, Faculty of Medicine,

Universiti Malaya,

50603 Kuala Lumpur, MalaysiaMarine algae are exposed to a combination of light and

oxygen that leads to the formation of free radicals and other

strong oxidizing agents (Dykens et al. 1992; Namiki 1990),

but the absence of oxidative damage in the structural

components (polyunsaturated fatty acids) of seaweeds

(Matsukawa et al. 1997) and their stability to oxidation

during storage (Ramarathnam et al. 1995) suggest their cells

have protective antioxidative defense systems (JiménezEscrig et al. 2001). Marine algae contain phloroglucinol

phenolics (phlorotannins) (Pavia and Aberg 1996) which are

probably good antioxidants (Ragan and Glombitza 1986),

since plant phenolics can behave as ROS scavengers, metal

chelators and enzyme modulators and prevent lipid peroxidation (Rodrigo and Bosco 2006). Polyphenols are reducing

agents, and together with other dietary reducing agents such

as vitamin C, E and carotenoids, referred to as antioxidants,

protect the body’s tissues against oxidative stress and

associated pathologies such as cancer, coronary heart disease

and inflammation (Tapiero et al. 2002).

The antioxidant activity of several seaweeds has been

published (Matsukawa et al. 1997; Anggadiredja et al.

1997; Jiménez-Escrig et al. 2001; Yan et al. 1998; Rupérez

et al. 2002; Nagai and Yukimoto 2003), but there are no

publications on the antioxidant activities of seaweeds from

Malaysian North Borneo (Sabah). Since marine algae are

rich source of dietary fiber, minerals, proteins and vitamins

(Yan et al. 1998), a documented antioxidant activity of

these seaweeds would elevate their value in the human diet

as food and pharmaceutical supplements.

Many authors have stressed the need to carry out more

than one type of antioxidant activity measurement to take

into account the various mechanisms of antioxidant action

(Frankel and Meyer 2000; Prior and Cao 1999), as no

single assay will accurately reflect all of the radical sources

or all antioxidants in a mixed or complex system (Prior et

al. 2005). In the present study, we evaluated the antioxidant

activities of seaweeds using trolox equivalent antioxidant

capacity (TEAC) and ferric reducing antioxidant power

(FRAP) assays. These two analytical methods are routinely

used to assess antioxidant activities in vitro. We also

estimated the total phenolic contents of these seaweeds

using the classical Folin–Ciocalteu reagent, and investigated the relationship between the total antioxidant capacities

and phenolic contents in the seaweed samples.

Materials and methods

ABTS (2,2 -azino-bis-(3ethylbenzothiozoline-6-sulfonic acid)′

diammonium salt), TPTZ (2,4,6-tri(2-pyridyl)-S-triazine),

potassium persulfate (K2S2O8), Folin-Ciocalteu reagent,

butylated hydroxytoluene (BHT) and quercetin were purchased from Sigma (USA). Trolox (6-hydroxy-2,5,7,8-

tetramethylchroman-2-carboxylic acid), a water analogue of

vitamin E, was purchased from Aldrich (USA). Phloroglucinol (trihydroxybenzen) was obtained from Fluka Chemicals (Switzerland) and Ferric chloride hexahydrate

(FeCl3

.6H2O) came from BDH (England). Sodium sulphate

anhydrous, methanol, diethyl ether, and hydrochloric acid

were from Merck (Germany). All other reagents were of

analytical grade.

Specimens of the eight seaweed species were collected

from the coastal areas of Sabah, Malaysia. Eucheuma cottonii

and E. spinosum were harvested from the Universiti

Malaysia Sabah farms in Bangi, Kudat (north coast of

Sabah). Caulerpa lentillifera and C. racemosa were collected from Semporna (east coast of Sabah) and Tanjung Aru

(west coast of Sabah) waters, respectively. Halymenia

durvillaei, Dictyota dichotoma, Sargassum polycystum and

Padina spp. were collected from Tanjung Aru waters (west

coast of Sabah). Fresh seaweeds were washed with distilled

water and their holdfasts and epiphytes removed. The fresh

seaweed was placed in a freezer (−20°C) immediately after

collection. All cleaned seaweeds were freeze-dried at −50°C

for 3 days and then ground to fine powder using a Waring

miller to pass through a 0.5-mm screen, and stored in airtight containers at −20°C.

Two extraction procedures were carried out according to

the modified method of Mohd Zin et al. (2002) and

Vairappan (2003). The procedures were as follows. First,

ground freeze-dried seaweed samples (10 g) were extracted

with 100 mL of methanol at room temperature for 72 h. The

samples were then filtered with Whatman filter paper no. 1

and the solvent removed under vacuum at 40°C using a

rotary evaporator to give a dark green viscous mass. This

extract is the methanolic dry extract. Second, ground

freeze-dried seaweed samples (10 g) were extracted with

100 mL of methanol at room temperature for 72 h. Samples

were filtered and the methanol solution was concentrated in

vacuo and partitioned between diethyl ether and water

(ratio 1:3). Diethyl ether was washed with water, dried over

anhydrous sodium sulphate and evaporated to leave a dark

green oil. This extract is the diethyl ether dry extract.

The methanol extract was used since it has shown to

give the highest antioxidant activity in many seaweed

species (Yan et al. 1999). Previous study had shown that, in

the case of methanol extraction, the content of polyphenols

at 72 h was higher than at 24 h (unpublished data),

therefore extraction was conducted for 72 h to maximize

the yield of polyphenols. According to Lapornik et al.

(2005), yield of polyphenols in alcohol extracts strongly

increases with a longer time of extraction. The dried

extracts were weighed and the yield extracts obtained were

calculated. The solvent extract that gave higher yield was

further subjected to TEAC and FRAP assays for comparison of their antioxidant activities.

368 J Appl Phycol (2008) 20:367–373TEAC assay

The seaweed extracts (1 mg dry extract diluted in 1 mL

methanol) antioxidant activities were measured using the

TEAC method as described by Re et al. (1999) with some

modifications. The reaction was carried out in a microtiter

plate. Briefly, ABTS

+

radical cation was generated by a

reaction of 7 mM ABTS with 2.45 mM potassium

persulfate. The reaction mixture was allowed to stand in

the dark for 16 h at room temperature. Working solution

was prepared by diluting 1 mL of ABTS stock solution

with 19 mL phosphate buffered saline (PBS, 5 mM, pH 7.4)

where 200 μL of this working solution was dispensed to

each well of microtitration plate. Addition of 20 μL diluted

methanolic extracts (20 μL of sample extract was diluted

with 60 μL of methanol) initiated the reaction and

absorbance was read after exactly 6 min. A microplate

reader was used to read the absorbance at 645 nm. Trolox, a

vitamin E analogue in the concentration of 0 to 2.5 mM,

was used as standard and calibration. BHT and quercetin

whose concentrations were 1 mg mL

−1

, respectively, were

used as positive controls. All measurements were performed in triplicate. The results are expressed as mM

Trolox.mg

−1

dry weight of extract.

FRAP assay

The seaweed extracts (1 mg dry extract diluted in 1 mL

methanol) antioxidant activities were measured using FRAP

assay according to the Benzie and Strain (1999) method with

some modifications. The reaction was carried out in a

microtiter plate. The antioxidant activity of the standards was

estimated by using the increase in absorbance caused by the

generated ferrous ion. The working FRAP reagent contained

300 mM acetate buffer (pH3.6), 10 mM TPTZ 40 mM HCl

and 20 mM FeCl3.6H2O in the ratio of 10:1:1, freshly

prepared and warmed to 37°C. Two hundred μL of this

working solution was dispensed to each well of the microtitration plate. Then, addition of 20 μL diluted methanol

extracts (20 μL of sample extract was diluted with 60 μL of

methanol) initiated the reaction and absorbance was read

after exactly 10 min. A microplate reader was used to read

the absorbance at 593 nm. Trolox, in the concentration of 0

to 1000 μM, was used as standard and for calibration. BHT

and quercetin whose concentrations were 1 mg/1mL respectively were used as positive controls. All measurements were

performed in triplicate. The results were expressed as μM

Trolox.mg

−1

dry weight of extract.

Total phenolic content

The total phenolic (TP) content of the extract was determined

according to the Folin–Ciocalteu method (Velioglu et al.

1998) using phloroglucinol (a basic structural unit of

phlorotannins) as a standard and expressing results as

phloroglucinol equivalents (PGE) (Jiménez-Escrig et al.

2001). A 1.0 mL-aliquot of sample was added to 1.5 mL of

deionized water and 0.5 mL of Folin–Ciocalteu reagent (10×

dilution), and the contents were mixed thoroughly. After 1

min, 1.0 mL of 20% sodium carbonate solution was added,

and the mixture was again mixed thoroughly. The controls

contain all the reaction reagents except the sample. After

30 min kept in the dark, the absorbance was measured at

750 nm, and compared to a phloroglucinol calibration curve.

Statistical analysis

All data are expressed as means ± standard deviation. Data

were analyzed using one-way analysis of variance (ANOVA)

followed by Duncan multiple range tests by using the SPSS

system version 11.5 for Windows. Pearson’s correlation test

was used to assess correlations between means. A significant

difference was considered at the level of p<0.05.

Results

Table 1 shows the extraction yields of the methanolic and

diethyl ether extracts on a dry weight basis. The methanolic

extracts showed higher yield as compared with the diethyl

ether extracts for all seaweed samples. The extraction yield

of the methanolic dry extracts ranged from 1.88% to

40.33% while the diethyl ether dry extracts ranged from

0.47% to 10.87%.

Table 2 shows the results of primary screening of

antioxidant activity of all methanolic extracts using TEAC

assay expressed as TEAC value. This value represented

mM Trolox equivalents mg

−1

dry extract. The antioxidant

activities of the seaweed samples ranged from 1.49±0.02 to

2.16±0.04 mM Trolox equivalents mg

−1

dry extract. The

sequence of antioxidant activity of the methanolic seaweed

extract determined by TEAC assay was as follows: C.

Table 1 Extraction yield of seaweeds methanolic and diethyl ether

extracts on dry weight basis

Seaweeds Division Yield (%) w/w

Methanol Diethyl ether

Eucheuma cottonii Rhodophyta 2.25 0.86

Eucheuma spinosum Rhodophyta 1.88 0.59

Halymenia durvillaei Rhodophyta 7.92 1.68

Caulerpa lentillifera Chlorophyta 30.86 5.74

Caulerpa racemosa Chlorophyta 26.70 7.36

Dictyota dichotoma Phaeophyta 40.33 10.87

Sargassum polycystum Phaeophyta 4.05 0.47

Padina spp. Phaeophyta 8.55 2.87

J Appl Phycol (2008) 20:367–373 369lentillifera > C. racemosa > S. polycystum > D. dichotoma >

H. durvillaei > E. cottonii > E. spinosum > Padina spp.

The reducing activity of the seaweed extracts as

determined by FRAP assay varied as seen in Table 2. The

sequence of antioxidant activity of the methanolic seaweed

extract determined by FRAP assay was as follows: C.

lentillifera > C. racemosa > D. dichotoma > Padina spp. >

S. polycystum > E. cottonii > H. durvillaei > E. spinosum.

Table 3 shows the brown and green seaweeds have higher

phenolic content compared with the red seaweeds. The

sequence of total phenolic content of the methanolic seaweed

extracts was as follows: S. polycystum > C. lentillifera > C.

racemosa > D. dichotoma > Padina spp. > E. cottonii > H.

durvillaei > E. spinosum.

Figure 1 shows a relationship between free radicalscavenging activity, as TEAC value, and the reducing

power, as FRAP value, of eight species of seaweed crude

methanolic extracts. The correlation gave R

2

=0.89 indicating strong correlation between TEAC and FRAP assays.

Figure 2 shows a relationship between FRAP and TP values

which gave a correlation of R

2

=0.96 indicating strong

correlation. Figure 3 shows a relationship between TEAC

and TP which gave a correlation of R

2

=0.56 indicating

good correlation but not as strong as the correlation

between FRAP and TP values.

Discussion

The results presented in Table 1 show that there was an

increase in yield with increased solvent polarity (polarity

index of methanol and diethyl ether are 5.1 and 2.8,

respectively), an indication that more polar compounds were

found in seaweed extracts. Variation in the yields of various

extracts is attributed to polarities of different compounds

present in the plants. Since methanolic extracts gave significantly higher yield than diethyl ether for all seaweeds, the

methanolic extracts (dry weight basis) were used and tested

for antioxidant activity using TEAC and FRAP assays.

There are many methods to determine antioxidant

capacity. These methods differ in terms of their assay

principles and experimental conditions; consequently, in

different methods antioxidants in particular have varying

contributions to total antioxidant potential (Cao and Prior

1998). In this study, the seaweed extracts antioxidant

activities were tested using two different assays, TEAC

and FRAP assays. These two methods represented different

mechanisms of antioxidant action. A sample possessing

TEAC free radical-scavenging activity indicated that its

mechanism of action was as a hydrogen donor and

terminated the oxidation process by converting free radicals

to more stable products, whereas a compound exhibiting a

positive result in the FRAP assay was an electron donor and

it terminated the oxidation chain reaction by reducing the

oxidized intermediates into the stable form (Tachakittirungrod

et al. 2007).

Table 3 Total phenolic (TP) contents of seaweeds methanolic dry

extracts expressed as phloroglucinol equivalents (PGE)

Seaweeds TP mg PGE /g dry extract

Eucheuma cottonii 22.50±2.78

d

Eucheuma spinosum 15.82±1.24

e

Halymenia durvillaei 18.90±1.03

de

Caulerpa lentillifera 42.85±1.22

ab

Caulerpa racemosa 40.36±1.05

b

Dictyota dichotoma 35.23±5.65

c

Sargassum polycystum 45.16±3.01

a

Padina spp. 33.11±3.96

c

Values are expressed as mean±standard deviation, n=3

Different superscript letters within a column indicate significant

differences between samples at the level of p<0.05

10

9

4

7

5

8

2

3

1

6

0.0

1.0

2.0

3.0

4.0

5.0

0.0 100.0 200.0 300.0 400.0 500.0 600.0 700.0

FRAP values (uM/mg)

TEAC values (mM/mg)

Fig. 1 Correlation of FRAP and TEAC values of seaweed methanolic

dry extracts from (1) Eucheuma cottonii, (2) Eucheuma spinosum, (3)

Halymenia durvellaei, (4) Caulerpa lentillifera, (5) Caulerpa racemosa, (6) Dictyota dichotoma, (7) Sargassum polycystum, (8) Padina

spp, (9) BHT and (10) Quercetin

Table 2 Antioxidant activities of seaweeds methanolic dry extracts

determined by TEAC and FRAP assays

Seaweeds TEAC

(mM.mg

−1

dry

extract)

FRAP

(μM.mg

−1

dry

extract)

Eucheuma cottonii 1.63±0.09

fg

225.00±11.31

e

Eucheuma spinosum 1.54±0.08

gh

153.97±8.07

f

Halymenia durvillaei 1.67±0.04

e

182.29±13.35

f

Caulerpa lentillifera 2.16±0.04

c

362.11±15.65

c

Caulerpa racemosa 2.01±0.04

d

355.36±20.65

c

Dictyota dichotoma 1.66±0.09

f

268.86±13.30

d

Sargassum polycystum 1.86±0.02

h

366.69±11.85

c

Padina spp. 1.49±0.07

h

251.43±14.07

de

Butylated

hydroxytoluene

3.84±0.06

a

615.71±29.73

a

Quercetin 3.65±0.08

b

557.36±19.99

b

Values are expressed as mean±sandard deviation, n=3

Different superscript letters within a column indicate significant

differences between samples at the level of p<0.05

370 J Appl Phycol (2008) 20:367–373The TEAC assay applied in this study was according to

the improved technique described by Re et al. (1999) for the

generation of ABTS

•-

which involves the direct production

of the blue/green ABTS

•-

chromophore though the reaction

between ABTS and K2S2O8. This method is applicable to the

study of both water-soluble and lipid-soluble antioxidants,

pure compounds and food extracts. According to Ragan and

Glombitza (1986), radical-scavenging capacity of seaweeds

methanol extracts might be mostly related to their phenolic

hydroxyl group. In the present study, the green seaweeds

Caulerpa lentillifera and C. racemosa had greater antioxidant activities compared to the brown and red seaweeds. The

positive controls, BHT and quercetin, showed extremely

high antioxidant activity (TEAC values were above 3.0)

while all the seaweed samples showed high antioxidant

activity (TEAC values were below 3.0 but above 1.0). None

of the seaweed samples showed moderate antioxidant

activity (TEAC values below 1.0 but above 0.5) and low

antioxidant activity (TEAC values below 0.5).

In the FRAP assay, antioxidants in the sample reduce Fe

3+

/

tripyridyltriazine complex (Fe

3+

-TPTZ), present in stoichiometric excess, to the blue-colored ferrous form (Fe

2+

-TPTZ).

The antioxidant potential is propotional to the combined

(total) ferric reducing/antioxidant power (FRAP value) of the

antioxidants in the sample (Benzie and Szeto 1999). The reducing power property indicates that the antioxidant compounds are electron donors and can reduce the oxidized

intermediates of the lipid peroxidation process, so that they

can act as primary and secondary antioxidants (Yen and Chen

1995). We found that the green seaweeds C. lentillifera and

C. racemosa and the brown seaweed Sargassum polycystum

were more reactive then the other seaweeds, which were

similar to the results obtained in the TEAC assay. The

positive controls, BHT and quercetin, showed significantly

higher antioxidant activity than all the seaweed samples.

In this study, strong positive correlation between TEAC

and FRAP assays (R

2

=0.89) indicated the compounds

present in the methanolic extracts of seaweeds capable of

reducing ABTS radical were also able to reduce ferric ions.

Pulido et al. (2000) reported, in general, that the ferric ion

reducing ability of antioxidant correlates with the results

from other methods used to estimate antioxidant capacity.

Similarly, Thaipong et al. (2006) also reported high

correlation between TEAC and FRAP assays.

The phenolic contents of these seaweeds were evaluated

using the Folin–Ciocalteu method. The variation of phenolic

content was quite large (Table 3). The brown seaweed S.

polycystum and green seaweed C. lentillifera showed

significantly higher phenolic content than all the red

seaweeds. Jiménez-Escrig et al. (2001) also reported similar

findings that brown seaweeds contained higher phenolic

content than the red seaweeds. In agreement with previous

studies (Nagai and Yukimoto 2003; Duan et al. 2006), there

was a significant correlation between antioxidant activity and

phenolic content of these eight species of seaweeds. Many

algal species contain polyphloroglucinol phenolics (phlorotannins) (Pavia and Aberg 1996; Nakamura et al. 1996) and

in this study the antioxidant activity of algae could be due to

these compounds. The phenolic content in the seaweed

extracts showed a much higher correlation with reducing

power (R

2

=0.96) than the radical-scavenging activity (R

2

=

0.56). The lower correlation between TEAC values and the

phenolic contents in the seaweed extracts indicated that not

only the phenolic compounds were involved in the antioxidant activity through this pathway but there might be some

effects involving other active compounds.

Conclusions

Methanol showed higher extraction yield than diethyl ether

indicating more polar compounds were found in these

2

3

1

8

5

4

7

6

0.0

10.0

20.0

30.0

40.0

50.0

0.0 0.5 1.0 1.5 2.0 2.5

TEAC values (mM/mg dry extract)

TP (mg PGE/g dry extract)

Fig. 3 Correlation of TEAC and TP content of seaweed methanolic

extracts from (1) Eucheuma cottonii, (2) Eucheuma spinosum, (3)

Halymenia durvellaei, (4) Caulerpa lentillifera, (5) Caulerpa racemosa, (6) Dicyota dichotoma, (7) Sargassum polycystum and (8)

Padina spp

7

4

5

6

8

1

3

2

0.0

10.0

20.0

30.0

40.0

50.0

0.0 50.0 100.0 150.0 200.0 250.0 300.0 350.0 400.0

FRAP values (uM/mg dry extract)

TP (mg PGE/g dry extract)

Fig. 2 Correlation of FRAP and TP content of seaweed methanolic

extracts from (1) Eucheuma cottonii, (2) Eucheuma spinosum, (3)

Halymenia durvellaei, (4) Caulerpa lentillifera, (5) Caulerpa racemosa, (6) Dictyota dichotoma, (7) Sargassum polycystum and (8)

Padina spp

J Appl Phycol (2008) 20:367–373 371seaweed extracts. The green seaweeds C. lentillifera and C.

racemosa and the brown seaweed S. polycystum showed

better radical-scavenging and reducing power ability, and

higher phenolic content, than the other seaweeds. These

seaweeds could be potential rich sources of natural antioxidants. The TEAC and FRAP assays showed positive and

significantly high correlation (R

2

=0.89). There was a strong

correlation (R

2

=0.96) between the reducing power and the

total phenolic content of the seaweedsmethanolic extracts

expressed as phloroglucinol equivalents. The present findings appear useful in leading to further study in the

identification and characterization of specific compounds

responsible for the relatively high antioxidant activities in

these seaweeds. These studies are now in progress.

Acknowledgements The authors would like to thank Borneo

Marine Research Institute at the Universiti Malaysia Sabah and Sabah

Fisheries Department for supplying the seaweeds, Dr. Charles

Vairappan for collection of seaweeds and technical assistance, and

Teh Ooi Kock of University Malaya for their technical assistance in

the antioxidant assays. This study was funded by the Ministry of

Science, Technology and Innovation of Malaysia.

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