Jurnal Ilmu dan Teknologi Kelautan Tropis, Vol. 5, No. 2, Hlm.

287-297, Desember 2013

CHEMICAL COMPOSITION AND ANTIOXIDANT ACTIVITY OF

TROPICAL BROWN ALGAE Padina australis FROM PRAMUKA ISLAND,
DISTRICT OF SERIBU ISLAND, INDONESIA

Joko Santoso1*, Fitriany Podungge1, and Heru Sumaryanto1

1
Department of Aquatic Products Technology, Faculty of Fisheries and Marine
Sciences, Bogor Agricultural University, Bogor.
*
E-mail: [email protected]

Abstract
The proximate composition, dietary fiber, and total phenol contents as well as
antioxidant activity of tropical brown alga Padina australis collected from the Pramuka
Island, District of Seribu Island, Indonesia during the rainy season of 2011 were
determined in order to evaluate their potential nutritional value and activity of natural
antioxidant compound. The content of ash, protein, and fat were 22.26, 10.76, and 4.17
g/100 g dry matter, respectively; whereas the amounts of soluble, insoluble, and total
dietary fibers were 8.4, 5.4, and 13.8 g/100 g, respectively. Methanol extract of P.
australis contained the highest total phenol of 246.1 mg GAE/1000 g dry sample. The
extract also had the highest activity on DPPH radical scavenging, measured by IC50 of
267.1 ppm. Both the total phenol and IC50 value extracts decreased in the following
order: methanol > ethyl acetate > n-hexane.

Keywords: antioxidant, dietary fiber, DPPH-scavenging, Padina australis, proximate

composition

I. INTRODUCTION consumer (Arasaki and Arasaki, 1983;

Nisizawa et al., 1987; Nisizawa, 2002).
Seaweeds or marine macroalgae Seaweeds are the richest source of
are potential renewable resource in the minerals (Ruperez, 2000; Norziah and
marine environment. Approximately Ching, 2000; Santoso et al., 2006) and
6000 species of seaweeds in the world rich in dietary fibers contents (Wong and
have been identified and are grouped into Cheung 2000, Santoso et al., 2002;
different classes i.e. green (Chlorophytes), Benjama and Masniyom, 2012). Seaweeds
brown (Phaeophytes) and red also contain fat and protein in low
(Rhodophytes) algae (Devi et al., 2011). concentration (Wong and Cheung, 2000;
Indonesia as an archipelagic country has a Sánshez-Machado et al., 2004; Denis et
large number of seaweeds. Siboga al., 2010). Moreover, seaweeds contain a
expedition (1899-1900) succeeded to wide range of bioactive compounds
collect 555 species of seaweeds from mainly polyphenols with potential
Indonesian territorial waters, and among antioxidant activity.
them, around 20-30 have been utilized by Several studies have been
local people as foodstuff or traditional conducted and revealed that extract of
medicine (Mubarak et al., 1991). green, brown, and red seaweeds had
From foodstuff viewpoint, sea- antioxidant activities, measured by several
weeds become an important part of the markers such as peroxide value, chelating
diet of many Asian countries, such as ion metal, and scavenging of radical
Japan, China, and Korea. Japan particu- (Santoso et al., 2004a; 2010; Devi et al.,
larly, is the most important seaweed

©Ikatan Sarjana Oseanologi Indonesia dan

Departemen Ilmu dan Teknologi Kelautan, FPIK-IPB 287
Chemical Composition and Antioxidant Activity...

2011; O’Sullivan et al., 2011; Meenakshi II. METHODS

et al., 2012). Polyphenols derived from
seaweeds may be more potent than 2.1. Location and Sampling
analogous polyphenols derived from Preparation
terrestrial plant sources due to presence of Fresh tropical brown alga P.
up to eight interconnected phenol rings australis samples were collected in
(Hemat, 2007). In addition, bioactive February 2011 from the intertidal region
compounds identified in seaweeds of Pramuka Island, Seribu Island District,
including alkaloids, terpenes, ascorbic Indonesia (Figure 1). Immediately the
acid, tocopherols, carotenoids and samples were placed in plastic bags
phlorotannins have demonstrated containing sea water in order to prevent
antioxidant activity within in vitro studies evaporation and transported to the
(Hu et al., 2008; Heo et al., 2009; Li et laboratory under refrigerated condition.
al., 2011). Then, the plant was washed thoroughly
The nutrient compositions and with tap water to remove all sand
bioactive compounds of seaweeds vary particles, epiphytes and other impurities.
according to the species, maturity, The samples were divided into two
environmental growth condition, and groups, specifically fresh and dried
seasonal period (Ito and Hori, 1989; samples for the analysis of the proximate
Mabeau and Fluerence, 1993; Ortiz et al., composition and the content of dietary
2006). In addition, the changes in fibers, and for extraction of antioxidant
ecological condition have an influence on compound, respectively. Dried sample
the synthesis of nutrient and non-nutrient forms were obtained after being dried
compounds, including antioxidant using sun rays for two days and grounded
compound (Stengel et al., 2011; Benjama in an electric mixer. The powder sample
and Masniyom, 2012). was then stored in refrigerator for further
Seaweeds grown in the tropical use.
climate such as Indonesia are exposed to
high level of light, temperature and 2.2. P. australis Extracts
desiccation that can lead to increase in The procedure of extraction was
producing reactive radical species. In conducted according to the previous
order to survive, seaweeds may produce research conducted by Santoso et al.
some bioactive compounds and may (2010) with slightly modification. The
change the content of nutrient and non- powder of P. australis was macerated in
nutrient. In this study, tropical brown alga each solvent i.e., methanol, ethyl acetate,
P. australis were collected from the coast or n-hexane at a ratio of 1:16 (w/v) for 48
of Pramuka Island, Seribu Islands District, h under dark condition. Then the
Indonesia, during a rainy season. extraction was filtered through glass
The objectives of the study were to funnel and Whatman no. 42 filter paper.
measure the proximate composition and Each filtrate was concentrated to dryness
dietary fiber profile, to determine the under reduced pressure at temperature of
phenolic content of tropical brown alga P. 40 oC using a rotary flash evaporator until
australis extracted in different solvent, become paste. Each crude extract in paste
and to evaluate the function of seaweed form was filled up by nitrogen gas to
components as an antioxidant source prevent decomposition of active
through analysis of radical-scavenging compound inside, then was kept at -20 oC
activity. until analysis.

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Figure 1. The map of research site in Pramuka Island, Seribu Island District

2.3. Proximate Analysis total dietary fiber was calculated by

The proximate analysis consisted of summing the contents of insoluble and
moisture, ash, fat and protein were soluble dietary fibers.
carried out according to the procedures of
the Association of Official Analytical 2.5. Determination of Total Phenolic
Chemists (AOAC, 1995). The content of Contents
carbohydrate was calculated by Total phenolic contents of each
subtracting the weights of moisture, ash, crude extracts from P. australis were
fat and protein from 100 g sample. determined by spectrophotometry using
Follin-Ciocalteu reagents (Yangthong et
2.4. Determination of Dietary Fiber al., 2009). Each extract of P. australis
Contents was diluted with ethanol, added with
Insoluble and soluble dietary fibers distillated water and Follin-Ciocalteu
were determined according to an reagents. The mixture was allowed to
enzymatic-gravimetric method referred to stand for 5 min and added with sodium
the research conducted by Suzuki et al. carbonate. Homogenized mixture was
(1996) and Santoso (2003). Water then incubated in the dark room for one
insoluble dietary fiber was obtained after hour. The resulting absorbance was
boiling in water with pancreatin enzyme measured by a spectrophotometer (UV-
and phosphate buffer, filtered off by a 1200 UV-VIS Spectrophotometer,
glass fiber filter with ethanol and acetone. Shimadzu, Kyoto, Japan) at a wavelength
Water soluble dietary fiber was of 725 nm. Phenolic content was
precipitated from the filtrate using expressed in milligram per gram of dry
ethanol. The final corrected amounts of weight samples based on a standard curve
both dietary fibers were calculated by of gallic acid (GA), which was expressed
subtracting the weights of ash and protein as milligrams per 1000 gram of gallic acid
from the each fiber precipitate; while the equivalent (GAE).

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Chemical Composition and Antioxidant Activity...

2.6. DPPH Radical-Scavenging Activity high. Among three nutrient compounds,

Antioxidant activity assay was ash (mineral) became the highest content
conducted through the ability of the in compared to protein and fat. In our
sample on reducing the stable free radical previous research, we determined the
DPPH according to the method described proximate composition of fresh P.
by Aranda et al. (2009) with minor australis collected in dry season. The
modifications. Each crude extract was content of ash, protein and fat were 32.54,
weighed and then was added to ethanol 8.97 and 4.73 g/100 g dry matter,
with a ratio of 1:1000. Extracts with respectively (Santoso et al., 2006). The
several concentrations with the addition of content of ash was higher in dry season,
DPPH solution were loaded into the whereas the content of protein was higher
micro-well plate. The mixture was in rainy season. The content of fat both in
homogenized and incubated at 37 °C for rainy and dry season was almost same.
30 minutes. The resulting absorbance was Benjama and Masniyom (2012) reported
measured by a microplate reader that the high ash content of G. fisheri and
(Microplate Reader 168-1130, Biorad, G. tenuisipitata also was found in summer
California USA) at a wavelength of 517 season, whereas the protein content was
nm. Regression equation was obtained found high in rainy season. However, the
from the relationship between sample high fat content of G. fisheri and G.
concentration and percentage inhibition of tenuisipitata were found in summer and
free radical activity. rainy seasons, respectively. These levels
varied depending on the species,
2.7. Statistical Analysis environmental growth condition and
All the data were presented as seasonal period (Ito and Hori, 1989;
mean ± standard deviations. Statistical Mabeau and Fluerence, 1993; Ortiz et al.,
analyses were performed using student’s t 2006).
test and one-way analysis of variance. Dietary fibers belong to the non-
Multiple comparisons of means were nutritional compounds as an important
conducted using the least significance dietary constituent, which possesses a
difference (LSD) test. All computations wide range of positive properties
were done by employing the statistical (Leontowicz et al., 2001). In this study,
software (SPSS version 16). the amounts of soluble, insoluble and total
dietary fibers of 8.4, 5.4 and 13.8 g/100 g
III. RESULTS AND DISCUSSION fresh weight, respectively (Figure 2). The
content of total dietary fiber of brown
3.1. Proximate Composition and algae P. australis, Sargassum polycystum
Dietary Fiber Contents and Turbinaria conoides were 9.6, 10.1
The fresh alga sample contained and 9.5 g/100 g fresh weight, respectively
moisture, ash, protein, fat and (Santoso et al., 2006). However, G.
carbohydrate of 90.56 g/100 fresh weight, fisheri contained total dietary fiber of 57.5
2.11 g/100 fresh weight, 1.02 g/100 fresh - 64.0 g/100 g dry weight and G.
weight, 0.40 g/100 fresh weight and 5.90 tenuisipitata had total dietary fiber of 56.6
g/100 fresh weight, respectively (Table 1). – 60.2 g/100 g dry weight (Benjama and
After converting them to 100 g dry matter Masniyom, 2012).
samples, their content were shown to be

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Table 1. Proximate composition of fresh brown alga P. australis

Values (g/100 g)
Composition
Fresh weight Dry matter
Moisture 90.56 ± 0.16
Ash 2.11 ± 0.17 22.26 ± 1.98
Protein 1.02 ± 0.04 10.76 ± 0.54
Fat 0.40 ± 0.01 4.17 ± 0.04
Carbohydrate (by difference) 5.90 ± 0.37 62.21 ± 3.34

Figure 2. The contents of soluble, insoluble and total dietary fiber of P. australis

The ratio of soluble dietary fiber and minerals (Yoshie et al., 2000),
and insoluble dietary fiber of P. australis binding of bile salts (Wang et al., 2001),
was 1.6. This value was higher in and lipid metabolism effect (Wang et al.,
compared to red algae G. fisheri (0.39 - 2002).
0.42) and G. tenuisipitata (0.35 – 0.45)
(Benjama and Masniyom 2012), since the 3.2. Total Phenol Contents
genus Gracilaria has high content of Phenolic compounds were
soluble dietary fiber as sulphated commonly found in plants and have been
galactants (Wong and Cheung 2000). reported to have several biological
Different with dietary fiber from activities including potential antioxidants
terrestrial plants, dietary fibers in and free radical scavengers apart from
seaweeds contain some acidic group such primary defense role (Soobratte et al.,
as sulphuric group; therefore they have 2005). Methanol extract of P. australis
different characteristics in contained the highest total phenol of 246.1
physicochemical and physiological mg GAE/1000 g dry sample, followed by
effects, such as water holding capacity extract of ethyl acetate and n-hexane with
(Suzuki et al., 1996; Wong and Cheung, values were 90.17 mg and 17.3 mg
2000), oil holding capacity (Wong and GAE/1000 g dry sample, respectively
Cheung, 2000), swelling capacity (Wong (Figure 3).
and Cheung, 2000), binding of vitamins

Jurnal Ilmu dan Teknologi Kelautan Tropis, Vol. 5, No. 2, Desember 2013 291
Chemical Composition and Antioxidant Activity...

Figure 3. Total phenol content of each extract of P. australis. Letters over each
column in the graph not sharing the same are significantly different (p<0.05)

Compared to ethanol extract of phenol content can vary quite

brown alga Sargassum pallidum, the total considerably depending on the variety of
phenol content was higher than P. seaweed.
australis in spite of different standard and
extraction method. Fractionated ethanol 3.3. DPPH Radical-Scavenging Activity
extract of S. pallidum with chloroform, Methanol extracts of P. australis
ethyl acetate and n-buthanol contained the had stronger ability to scavenge DPPH
total phenol of 1.80, 11.54 and 12.12 mg radical in compared to others (Figure 4).
CHA/g extract (CHA = chlorogenic acid) IC50 value is defined as the concentration
(Ye et al., 2009). The levels of phenols in of substrate that can reduce 50% activity
the methanol extract of brown algae of DPPH radical. The IC50 value results
ranged from 1.5 mg GAE/g dry weight in decreased in the following order:
Laminaria hyperborea to 4.5 mg methanol > ethyl acetate > n-hexane; with
GAE/gdry weight in Ascophyllum values were 267.1, 1160.2 and 1629.5
nodosum (O’Sullivan et al., 2011). ppm, respectively.
Ganesan et al. (2008) observed methanol The effects of antioxidants on
extract of red seaweeds Eucheuma DPPH radical scavenging is thought to be
cottonii and Gracilaria edulis contained due to hydrogen donating ability. When a
total phenol of 1.5 and 4.1 mg GAE/g dry DPPH solution is mixed with a substrate
weight, respectively. Chandini et al. as hydrogen atom donor, a stable non-
(2008) on the other hand observed lower radical form of DPPH is obtained with
levels of phenols in the aqueous fractions simultaneous change of the violet color to
of Sargassum marginatum and Turbinaria pale yellow (Molyneux, 2004). Hence,
conoides which contained of 0.29 and DPPH has been used extensively as a free
0.86 mg GAE/g on a dry weight basis, radical to evaluate reducing substances
respectively. Furthermore, methanol and is a useful reagent for investigating
extract of T. conoides contained total the free radical scavenging activities of
phenol of 1.23 mg GAE/g (Devi et al., compound (Duan et al., 2006).
2011). Chew et al. (2008) reported that

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Figure 4. DPPH radical-scavenging activity of each extract of P. australis measured

by IC50. Letters over each column in the graph not sharing the same are
significantly different (p<0.05)

Based on IC50 value, the only contained total phenol of 2.5 mg

antioxidant compound are classified as GAE/g dry weight; however, it had
follows: very powerful antioxidant when highest activity on reducing a ferric
the IC50 values less than 0.05 mg/mL, oxidant to a ferrous complex by electron-
strong antioxidant if the value of IC50 transfer with value was 109.8 µM ascorbic
between 0.005 to 0.10 mg/mL, acid.
intermediate and weak when the IC50 Bioactive compounds identified in
values ranged from 0.10 to 0.15 mg/mL seaweeds including alkaloids, terpenes,
and from 0.15 to 0.20 mg/mL, ascorbic acid, tocopherols, carotenoids,
respectively (Molyneux, 2004). and phlorotannins (Hu et al., 2008; Heo et
According to the classification, the highest al., 2009; Li et al., 2011). The antioxidant
value of methanol extract belongs to weak activity of polyphenols depends on their
activity. nature (i.e. phenolic acids,
There was a positive correlation hydroxycinnamic acids, flavonoids, etc.)
between total phenol content and and chemical structure (mono or
antioxidant activity determined by DPPH dihydroxylation, etc.) (Cuvelier et al.,
radical scavenging. Devi et al. (2011) 1992; Pulido et al., 2000). Furthermore,
reported that methanol extract of T. Santoso (2003) and Santoso et al. (2004b)
conoides contained the highest number of succeeded to identify several active
phenolic compound, exhibited higher compounds from seaweed namely
radical scavenging activity. Similar result gallocatechin, epigallocatechin, catechin,
was reported by O’Sullivan et al. (2011) epicatechin, epigallocatechin gallate,
that methanol extract of A. nodosum had gallocatechin gallate, epicatechin gallate,
total phenol content of 4.5 mg GAE/g dry catechin gallate, and catechol.
weight also had highest activity on DPPH- There are different mechanisms of
radical scavenging of 25%. In contrary the antioxidant defense system, i.e. (1)
methanol extract of Fucus vesiculosus scavenging of oxygen and hydroxyl

Jurnal Ilmu dan Teknologi Kelautan Tropis, Vol. 5, No. 2, Desember 2013 293
Chemical Composition and Antioxidant Activity...

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