International Journal of Food Properties, 15:1182–1189, 2012
Copyright © Taylor & Francis Group, LLC
ISSN: 1094-2912 print / 1532-2386 online
DOI: 10.1080/10942912.2010.517341
DETERMINATION OF THE ANTI-OXIDATIVE CAPACITY
AND BIOACTIVE COMPOUNDS IN GREEN SEAWEED
ULVA RIGIDA C. AGARDH
Gamze Yildiz, Serap Celikler, Ozgur Vatan, and Sukran Dere
Biology Department, Science and Arts Faculty, Uludag University, Bursa, Turkey
There is an increasing demand for natural antioxidant molecules in order to replace the
synthetic additives in the food industry. Therefore, Ulva rigida C. Agardh was analyzed to
determine its bioactive components, including the total phenolic content, antioxidant capac-
ity (lipid and water-soluble), vitamins (A, E, and C), protein, carbohydrate, and pigments.
As a result, Ulva rigida showed a high total phenolic, vitamin E, and total carotene content.
Hence, U. rigida could be considered as a plant possessing natural antioxidant molecules
and might be useful for the food industry. U. rigida can also be used for curing diseases
arising from oxidative deterioration.
Keywords: Antioxidant molecules, Pigments, Total phenolic content, Ulva rigida,
Vitamins.
INTRODUCTION
Reactive oxygen species (ROS), such as hydrogen peroxide, superoxide radicals,
hydroxyl radicals, and singled oxygen, are physiological metabolites formed during aer-
obic life as a result of the metabolism of oxygen. ROS are toxic to cells. Excessive
production of such molecular compounds can cause damage to proteins, lipids, DNA, and
cell membranes. This damage can induce different kinds of diseases in the human body,
such as atherosclerosis, rheumatoid arthritis, diabetes, muscular dystrophy, pulmonary
dysfunction, mycocardial infarction, Alzheimer’s disease, and some types of cancer.[1]
ROS is scavenged by antioxidant molecules, such as ascorbate, glutathione, and toco-
pherol, and by enzymes, such as superoxide dismutase, catalase, ascorbate peroxidase,
and glutathione reductase.[2] The antioxidant molecules are essential for the body system.
Many researchers have reported that plant-derived antioxidants have a beneficial effect on
human health.[3,4] The presence of antioxidant compounds in the human diet can be helpful
to protect us from these diseases. Over the past several decades, seaweeds and their extracts
have been demonstrated to have strong antioxidant activity and they have been studied to
produce antioxidant compounds.[5]
The increasing demand for natural antioxidant compounds results in the search for
new sources of natural antioxidant molecules. Furthermore, Ulva rigida is used for human
nutrition. Because of the potential benefits listed above, there is a great interest in studying
and determining the amount of antioxidant components in this seaweed. In addition, reports
Received 20 May 2010; accepted 15 August 2010.
Address correspondence to Gamze Yildiz, Biology Department, Science and Arts Faculty, Uludag
University, Bursa 16059, Turkey. E-mail: gamze@uludag.edu.tr
1182
BIOACTIVE CONTENTS OF ULVA RIGIDA 1183
on the antioxidant properties of seaweed extracts from Turkey are very limited. Therefore,
in this study total phenolic content, total antioxidant capacity (in soluble of lipid and water),
total protein, total carbohydrate, vitamins (A, C, and E), and pigments (chlorophyll-a,
chlorophyll-b, and carotene) contents were determined in order to identify new sources
of natural antioxidant molecules.
MATERIALS AND METHODS
Collection of Samples
Ulva rigida was freshly collected from the Marmara Sea coast of Turkey during
November 2007. Samples were kept in seawater until they arrived at the laboratory. The
samples were immediately washed with top and distilled water to remove epiphytes, salt,
and dirty particles after arrival. The clean algae were frozen and stored at -20◦C. For the
analyses, the frozen samples were used.
Chemicals
Folin-Ciocalteau reagent (F-9252), gallic acid (G-7384), and alfa-tocopherol (T-
3251), L-ascorbic acid were purchased from Sigma Chemical Co. (St. Louis, MO,
USA). 2,6-Dichlorophenol indophenol, trichloroacetic acid were obtained from Merck
(Darmstadt, Germany). All other solvents and chemicals were of analytical grade.
Total Phenolic Content
Frozen samples were extracted with methanol for the total phenol analysis. Phenolic
contents were measured using Folin Ciocalteu’s method as described by Taga et al.[6] An
aliquot of 100 µl of the sample was mixed with 2 ml of 2% Na2CO3 and allowed to stand
for 2 min at room temperature. After incubation, 100 µl of 50% Folin Ciocalteu’s phenol
reagent was added, and the reaction mixture was mixed thoroughly and allowed to stand for
30 min at room temperature in the dark. The absorbance was measured at 720 nm and total
phenolic content was calculated with a Gallic acid standard and expressed as mg Gallic
acid equivalent per 100 gram of fresh tissue.
Determination of Lipid-Soluble Antioxidant Capacity
Frozen samples were homogenized with hexane and shaken for 1 h at 4◦C in the
dark. After centrifugation at 6000 g for 10 min, the supernatant was transferred to new
tubes. Samples of hexanic extracts (200 µl) were placed in Eppendorf tubes, dried out, and
re-dissolved in the same volume of ethanol. These ethanolic solutions were supplemented
with 1 ml of phosphomolybdenum reagent (32 mM sodium phosphate, 4 mM ammonium
molybdate, 0.6 M sulfuric acid) and incubated at 95◦C for 90 min. Finally, the absorbance
at 695 nm was measured. Lipid-soluble antioxidant capacity was expressed as equivalents
of α-tocopherol in micromoles of α-tocopherol per gram of fresh tissue.[7]
Determination of Water-Soluble Antioxidant Capacity
Frozen samples of water extracts (200 µl) were supplemented with 1 ml of phos-
phomolybdenum reagent and incubated at 95◦C for 90 min. Finally, the absorbance at
1184 YILDIZ ET AL.
695 nm was measured. Water-soluble antioxidant capacity was expressed as the equivalent
of L-ascorbic acid in micromoles of L-ascorbic acid per gram of fresh tissue.[7]
Determination of Vitamins E, C, and A
Vitamin E content was determined by using a method described by Prieto et al.[7]
Hexanic extract of frozen algal tissues (0.1 ml) was mixed with 1 ml of phosphomolyb-
denum reagent solution and incubated at 37◦C for 90 min with vigorous shaking. The
absorbance was measured at 695 nm. Vitamin E content was expressed as α-tocopherol
equivalents per 100 grams of fresh tissue. Ascorbic acid concentrations were deter-
mined by the titrimetic Association of Official Analytical Chemists (AOAC) method
No. 967.21. Frozen samples were homogenized with metaphosphoric acid solution and
then centrifuged at 6000 g for 10 min. Supernatants were titrated with 2,6-dichlorophenol
indophenol as a titrant.[8] Vitamin C content was expressed as mg L-ascorbic acid per
100 grams of fresh tissue. Vitamin A content was determined and calculated by using a
method described by Rutkowski and Grzegorczyk.[9] Frozen samples were extracted with
hexane. An aliquot of 1 ml of hexanic extract and 1 ml of KOH solutions were mixed
in a vortex for 1 min and incubated in a water bath at 60◦C for 20 min. Cooled samples
were mixed with 1 ml of xylene and centrifuged at 1500 g for 10 min. Absorbances of
the organic phase were determined both before and after irration with UV light at 335 nm
against xylene.
Determination of Total Soluble Carbohydrate and Protein
Total soluble carbohydrate was assayed by the anthrone-sulphuric acid method,
which involved extraction with 15% trichloroacetic acid.[10] The absorbance was measured
at 620 nm.
Total protein content was determined spectrophotometrically at 595 nm and con-
centrations were calculated by comparing with a calibration curve of bovine serum
albumin.[11]
Determination of Pigments (Chlorophyll-a, Chlorophyll-b, and Total
Carotene)
Chl a and Chl b contents for Chlorophyta were determined in accordance with the
Jeffrey and Humphrey method by using 90% acetone as solvent.[12] Car was determined
according to the Lichtentaler and Wellburn method by using 90% acetone.[13]
Statistical Analysis
Three samples were prepared for each experiment. The data were presented as
mean ± standard deviation.
RESULTS AND DISCUSSION
Total Phenolic Content
Using the Folin Ciocalteu method phenolic content of Ulva rigida was investi-
gated and expressed as mg gallic acid 100 g−1 FW (Table 1). Phenolic compounds are
BIOACTIVE CONTENTS OF ULVA RIGIDA 1185
Table 1 The contents of bioactive molecules and antioxidant capacity of Ulva rigida based on fresh weight
(Mean ± SD), (n = 3).
Ulva rigida C. Agardh
CALT (µmol α-tocopherol g−1) 130.91± 24.56
CAHT (µmol L-ascorbic acid g−1) 375.59± 61.63
Vitamin E (mg α-tocopherol 100 g−1) 147.00± 0.28
Vitamin A (µM) 0.91± 0.47
Vitamin C (mg L-ascorbic acid 100 g−1) 46.00± 0.17
Total phenol (mg gallic acid 100 g−1) 73.00± 0.13
Total protein (%) 52.33± 4.51
Total carbohydrate (%) 34.98± 20.39
Chlorophyll-a (mg 100 g−1) 51.13± 25.69
Chlorophyll-b (mg 100 g−1) 43.40± 19.32
Total carotene (mg 100 g−1) 9.67± 3.18
CAHT: Water soluble antioxidant capacity; CALT: lipid soluble antioxidant capacity.
Table 2 Total phenolic content of Ulva rigidaa and of some other seaweedsb.
Seaweeds Total phenolic (mg gallic acid g−1)
Ulva rigida 0.73± 0.13
Codium fragile 0.27± 0.02
Dictyopteris divaricata 0.96± 0.01
Scytosiphon lomentaria 0.52± 0.01
Gracilaria gracilis 0.10± 0.00
Ceramium kondoi 0.44± 0.01
aMean values, n = 3, fresh weight basis.
bSource: Zhang et al.[15] Values are based on dry weight.
commonly found in plants and have been reported to have several biological activities
including the antioxidant activity. The major part of antioxidant molecules are polypheno-
lic compounds.[14] Therefore, a number of studies have focused on the biological activities
of phenolic compounds.
In this study, the green algae Ulva rigida was found to contain a total phenolic
compound of 73 mg gallic acid 100 g−1 FW. This value was compared with the corre-
sponding data for several seaweeds, which was reported by Zhang et al.[15] They are also
included in Table 2. Table 2 shows that the amount of total phenolic content of U. rigida
was relatively higher than other seaweeds. Phenolic compounds have been highly prized
for their important dietary roles as antioxidant molecules and chemo-preventive agents.[16]
Celikler et al. have also reported that Ulva rigida have strong antigenotoxic activity in
human lymphocytes in vitro[17] and antihyperglicemic effect in in vivo.[18]
Total Antioxidant Capacity (Water and Lipid-Soluble)
Total water-soluble and lipid-soluble antioxidant capacity of U. rigida is presented
in Table 1. Mohamed et al.[19] observed the highest levels of water-soluble antioxidant
capacity of 277.7 µmol L-ascorbic acid g−1 in wheat germ and the highest level of
lipid-soluble antioxidant capacity of 118.5 µmol α-tocopherol g−1 in chili pepper seeds.
Total water and lipid-soluble antioxidant capacity of U. rigida (375.59 µmol L-ascorbic
acid g−1 FW and 130.91 µmol α-tocopherol g−1 FW, respectively) is relatively higher
1186 YILDIZ ET AL.
when compared with these plants. The positive correlation between polyphenolic content
of algae and its antioxidant activity is well documented.[20] In this study, U. rigida showed
a remarkable antioxidant capacity. Therefore, we think that this might be a result of its
high polyphenolic content.
Vitamin E, C, and A Contents
Vitamin E is the major lipid-soluble antioxidant responsible for protecting the
polyunsaturated fatty acids in membranes against lipid peroxidation, free radicals, and sin-
glet oxygen species.[21] In addition, α-tocopherol is the most common form of Vitamin
E present in nature and it is the most biologically active form. Ching and Mohamed[22]
investigated α-tocopherol content in 62 edible tropical plants. In their study, the high-
est α-tocopherol content was found to be 79.65 mg α-tocopherol 100 g−1 in Sauropus
androgynus. According to our data, Ulva rigida has a remarkably higher value (147 mg
α-tocopherol 100 g−1 FW) than that of Sauropus androgynus. High content of Vitamin E
in U. rigida is important because of its potential role in the prevention of heart disease and
cancer.[23]
Except for antioxidant properties of Vitamin E, recently other biological activities
have been reported, such as the regulation of cellular signaling and gene activity, modula-
tion of immune function, and induction of apoptosis.[24] Ascorbic acid, also referred to as
L-ascorbic acid or vitamin C, is a water-soluble vitamin and it is largely used in therapy
for anti-infections in cells.[25] The content of vitamin C in U. rigida is 46 mg of ascorbic
acid 100 g−1 FW and this value is higher than the values in Gracilaria changgi[26] and
some of the consumable vegetables.[27] The content of vitamin A in U. rigida (0.91 µM) is
presented in Table 1. Vitamin A or retinol is an essential nutrient for humans and animals
since it cannot be synthesized within the body. A deficiency of vitamin A can be lead to a
number of health problems.
Total Carbohydrate and Protein Contents
Marine plants characteristically contain sulfated polysaccharides that are not found
in land plants. In recent years, sulfated polysaccharides from marine algae have been
reported to have antioxidant activity.[28] Their activity depends on several structural
parameters, such as the degree of sulfation, the molecular weight, and the sulfa-
tion positions. In this study, U. rigida showed high carbohydrate content (Table 1).
However, Ortiz et al.[29] found that carbohydrate content of U. lactuca was 61.5%,
which is higher than our result (34.98%). The mean protein content found in this study
is in agreement with values reported for various macroalgae.[30] U. rigida showed a
high protein content (52.33%) similar to traditional high protein plant sources.[26,29]
This high protein content of U. rigida makes up a valuable source for human
nutrition.
Pigments (Chlorophyll-a, Chlorophyll-b, and Total Carotene)
Chlorophyll-a is the main green pigment found in most algae. It is one of the signif-
icant biomolecules that is lately being studied for its antioxidant properties. Its absorption
by human intestinal cells supports its potential importance for human health.[31] The sec-
ond kind of chlorophyll is chlorophyll-b, which occurs only in green algae and the plants.
BIOACTIVE CONTENTS OF ULVA RIGIDA 1187
U. rigida also contains excessive levels of both chlorophyll-a (51.13 mg 100 g−1 FW) and
chlorophyll-b (43.40 mg 100 g−1 FW, Table 1).
Carotene, especially β-carotene, is an important nutrient with provitamin A value.
The protective role of carotenoids is based on the effective quenching and prevention of
singlet oxygen formation. In our study, U. rigida showed higher total carotene content
(9.67 mg 100 g−1 FW) when it is compared with green chilies (2410 µg 100 g−1), tomato
(3090 µg 100 g−1), and maize (1782 µg 100 g−1).[32] Many investigators have studied the
relation between dietary carotenoid intake and health. Anti-ageing effects of carotenoids
were also demonstrated by Cutler.[33]
CONCLUSIONS
Ulva rigida, a green algae found in Turkey, was analyzed in terms of its bioactive
components. In this study, the nutritional value and biologically active compounds were
compared with the values previously found in several plants. According to this study, it can
be concluded that seaweeds can be utilized as a source of natural antioxidant compounds.
They can enhance the antioxidant defense system of the human body. The present study
appears to be useful for leading the development of therapeutic products to protect people
against certain diseases. The results of this study may be well used by other researchers
to analyze the characterization of the biologically active molecules that are responsible for
the antioxidant activity in U. rigida.
ACKNOWLEDGMENTS
This investigation was supported by a grant from The Scientific and Technical Research
Council of Turkey, Ankara (TUBITAK, project no. 107T279 (TBAG/HD-304).
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