Accepted Manuscript

Isolation and Antioxidant Capacity of Fucoidan from Selected Malaysian Seaweeds

Seng Joe Lim, Wan Mustapha Wan Aida, Maskat Mohamad Yusof, Said Mamot,
Jokiman Ropien, Diah Mazita Mohd

PII: S0268-005X(14)00080-0
DOI: 10.1016/[Link].2014.03.007
Reference: FOOHYD 2534

To appear in: Food Hydrocolloids

Received Date: 3 January 2013

Revised Date: 5 March 2014
Accepted Date: 10 March 2014

Please cite this article as: Lim, S.J., Wan Aida, W.M., Mohamad Yusof, M., Mamot, S., Ropien, J.,
Mazita Mohd, D., Isolation and Antioxidant Capacity of Fucoidan from Selected Malaysian Seaweeds,
Food Hydrocolloids (2014), doi: 10.1016/[Link].2014.03.007.

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 ACCEPTED MANUSCRIPT

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Extraction
•2 CaCl2 (aq)
Antioxidant study

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Screening Identification,
•85°C, 24h, 6 times confirmation •Total phenolic
•Sargassum binderi content
•Padina sp. Isolation and purity of
•Free-radical
•Gracilaria sp. •Precipitation (CTAB) fucoidan
•Reducing power

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•Eucheuma cottonni •Exchanged (NaI EtOH) •HPAEC •Superoxide anion
•Dialysis
•Eucheuma spinosum •ATR-FTIR •Hydroxyl radical

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•Lyophilization

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1 Isolation and Antioxidant Capacity of Fucoidan from Selected Malaysian Seaweeds

2 Lim Seng Joea, Wan Aida Wan Mustaphaa*, Mohamad Yusof Maskata, Mamot Saida,

3 Ropien Jokimanb, Mazita Mohd Diahb

a
4 School of Chemical Science and Food Technology, Faculty of Science and Technology,

5 Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia

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b
6 Cosmetics and Natural Products Programme, Industrial Biotechnology Research

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7 Centre, SIRIM Berhad, No. 1, Persiaran Dato' Menteri, Seksyen 2,

8 Peti Surat 7035, 40911 Shah Alam, Selangor, Malaysia

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9 *Corresponding author: wawm@[Link] (email)

10 603-89213870 (tel), 603-89213232 (fax)

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12 ABSTRACT
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13 The objective of this research was to screen and isolate fucoidan from Malaysian seaweeds

14 and subsequently determine its antioxidant capacity. A screening test employing a

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15 colorimetric method was conducted on five types of Malaysian brown and red seaweeds. It
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16 was found that Sargassum binderi contained the highest fucoidan content (6.16 ± 0.08%).

17 Thus, fucoidan was isolated from Sargassum binderi (yield 7.5%, purity 89.63%) for the
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18 determination of its antioxidant capacity. The isolated fucoidan was identified using high
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19 performance anion exchange chromatography (HPAEC) and attenuated total reflectance

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20 Fourier transform infra-red (ATR-FTIR) spectroscopy. The antioxidant assays performed

21 were total phenolic content (TPC), free-radical scavenging activity (DPPH), reducing power,

22 superoxide anion scavenging activity (SOA) and hydroxyl radical scavenging activity (·OH).

23 The antioxidant capacity of the extracted fucoidan (Fsar) was compared with those of a

24 commercial food-grade fucoidan (Fysk) and of BHA, BHT and ascorbic acid. All the

25 antioxidant assays performed showed either Fsar has significantly higher (p<0.05) or do not
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26 differ significantly (p>0.05) in activities compared to that of Fysk. At the same time, both Fsar

27 and Fysk showed significant (p<0.05) antioxidant capacity in terms of superoxide anion and

28 hydroxyl radical scavenging activities compared to those of the synthetic antioxidants. This

29 shows that Fsar has the potential to be commercialised as a functional food product or as

30 bioingredients with high antioxidative properties.

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31

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32 Keywords: Antioxidant, attenuated total reflectance Fourier transform infra-red (ATR-

33 FTIR), fucoidan, Malaysian seaweeds, Sargassum binderi.

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34

35 1. Introduction

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Marine resources, in particular seaweeds, have drawn significant attention in recent
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37 years in the search for bioactive compounds to develop as functional food and nutraceutical
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38 products (Souza et al., 2012). The seaweed industry has an annual global value of USD 5.5-6

39 billion and is mainly used for food (USD 5 billion), phycocolloids (hydrocolloids), fertiliser,
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40 animal feed additives, cosmetics and medicines (Phang, 2010). The Food and Agricultural
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41 Organization of the United Nations (FAO) (2012) reported that in 2010, worldwide aquatic

42 plant production reached 19 million tonnes, worth USD 5.56 billion. In Malaysia, aquatic
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43 plant production has developed enormously over the past few years, from 18,863 tonnes
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44 worth USD 1.99 million in 2001 to 207,892 tonnes worth USD 17.44 million in 2010. The
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45 value of aquatic plants in Malaysia has also increased tremendously, from USD 56.78 per

46 tonne in 2009 to USD 83.91 per tonne in 2010 (FAO, 2012). Malaysian national frameworks

47 and strategies have placed a significant emphasis on seaweed production. This includes the

48 Ninth Malaysia Plan (2006-2010), the Third National Agricultural Policy (1998-2010), the

49 recent adoption of the National Aquaculture Centre and the Malaysian Budget 2010, with

50 seaweed being mentioned specifically as one of the most important food farming
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51 commodities for the country (Kaur & Ang, 2009). The abundant supply of seaweeds in

52 Malaysia offers great opportunities for producing and extracting several functional

53 ingredients, such as fucoidan, alginate, agar and carrageenan.

54 Seaweeds are marine macroalgae that can be classified into the Chlorophyta (green

55 seaweeds), Rhodophyta (red seaweeds) and Phaeophyta (brown seaweeds) (Phang, 2010).

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56 Fucoidan is a type of glyconutrient and one of the main polysaccharides of brown seaweeds.

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57 According to Bilan et al. (2010) and Zvyagintseva et al. (1999), fucoidan is the family of

58 sulphated homo- and heteropolysaccharides composed mainly of α-(1→2)-, α-(1→3)- and/or

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59 α-(1→4)-linked α-L-fucosepyranose residues. The main monomer in fucoidan is fucose, one

60 of the eight essential biological sugars. It also contains galactose, mannose, xylose and

61
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glucuronic acid residues (Bilan et al. 2010). Fucoidan is a nontoxic polyelectrolyte that
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62 possesses various pharmacological activities, i.e. antioxidant, antibacterial, antiviral,
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63 antitumor and anticoagulant activities, and it has also been developed as a specialised type of

64 nutraceutical and food supplement (Bilan et al., 2002; Zvyagintseva et al., 1999). Brown
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65 seaweed extracts (fucoidan) and seaweed polysaccharides are reported to have strong
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66 antioxidative properties and have high potential to be applied in pharmaceuticals,

67 nutraceuticals, cosmeceuticals and functional food (Barahona et al., 2011; Li et al. 2008;
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68 Wijesinghe & Jeon, 2012). Due to concerns regarding the carcinogenic and toxic effects of
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69 synthetic antioxidants, the demand for natural antioxidants has increased (Yangthong et al.,
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70 2009).

71 Despite the great number of studies on fucoidan, none has investigated fucoidan from

72 Malaysian seaweeds. It is well known that the biological activities of fucoidan vary according

73 to the season, the age of the population, species and geographic location (Rioux et al., 2007;

74 Zvyagintseva et al., 2003). The abundant supply of Malaysian seaweeds and the large variety

75 of their species make it possible to exploit Malaysian seaweeds for fucoidan production. In
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76 this research, five types of Malaysian seaweed were screened using a colorimetric method to

77 determine which type had the highest fucoidan content; based on this analysis, Sargassum

78 binderi was then chosen for further analysis. Fucoidan was extracted from Sargassum binderi

79 and was then isolated and lyophilised to obtain fucoidan in solid form (Fsar). The isolated

80 fucoidan was then tested for its antioxidative activities, in which commercial food-grade

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81 fucoidan (Fysk) and synthetic antioxidants (BHA, BHT and ascorbic acid) were used as

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82 comparisons.

83

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84 2. Materials and methods

85 2.1 Materials and samples

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Brown seaweeds (Sargassum binderi and Padina sp.) and red seaweeds (Gracilaria
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87 sp., Eucheuma cottonii and Eucheuma spinosum) were obtained from Semporna, Sabah,
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88 Malaysia. All the seaweeds were obtained in November 2010. Commercial food-grade

89 fucoidan with 92% purity from Okinawa mozuku was supplied by Yaizu Suisankagaku
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90 Industry Co., Ltd., Yaizu City, Japan (Fysk). Fucoidan standard (from Fucus vesiculosus) was
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91 obtained from Sigma, UK (Fsig). Synthetic antioxidant butylated hydroxyanisole (BHT)

92 (Merck, Germany), butylated hydroxytoluene (BHT) (Sigma, UK) and ascorbic acid (Fluka,
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93 UK) were also used. All other chemicals were of analytical grade and purchased from Sigma,
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94 unless otherwise stated.

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95

96 2.2 Sample preparation

97 Fresh seaweed samples were washed to remove salt and impurities and dried at 40°C

98 in an oven (Protech, Texas, USA) for 72 h. They were then cut into small pieces with a knife

99 mill before being ground into powder form using a high-speed grinder (Kuao Fung Electronic

100 and Machine, Taiwan). The samples were kept at 4°C until use.
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101

102 2.3 Screening of fucoidan content

103 Fucoidan was extracted twice from 150 mg of all five types of seaweed samples

104 (Brown seaweeds – Sargassum binderi and Padina sp.; and red seaweeds – Gracilaria sp.,

105 Eucheuma cottonii and Eucheuma spinosum) using 50 ml of 0.2 N HCl at 70°C for 1 h with

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106 mechanical stirring (Usov et al., 2001). The residues were separated through centrifugation

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107 (Eppendorf Centrifuge 5810R, at 3500 g, 5 min), and the supernatants were pooled and

108 brought to 100 ml. Aliquots of 1 ml were pipetted into test tubes placed in an ice-water bath,

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109 and 4.5 ml diluted sulphuric acid was added (1:6, H2O:H2SO4). After 1 min of cooling in an

110 ice-water bath, the test tubes were placed into a boiling-water bath for exactly 10 min. The

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aliquots were allowed to cool to room temperature before adding 0.1 ml of 3% L-cysteine,
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112 and the solutions were mixed and allowed to stand for 30 min. The absorbance at 396 nm and
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113 427 nm was measured in a 96-well plate using a BioTek Epoch microplate spectrophotometer

114 (Vermont, USA) (Hellebust & Craigie, 1978). A calibration curve within the 0.0025% to
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115 0.015% range of fucoidan (Fysk) was used to determine the fucoidan content in the samples.
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116 An n=3 replication was conducted in the screening process.

117
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118 2.4 Isolation of fucoidan

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119 The isolation of fucoidan was performed according to the procedures reported by
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120 Bilan et al. (2004). The ground Sargassum binderi (50.0 g) was treated with 200 ml of

121 MeOH:CHCl3:H2O (4:2:1) solution at room temperature with stirring for 24 h to

122 extract/remove lipids, coloured matter and low molecular weight components from the

123 sample. The suspension was then filtered, and the residue (ground Sargassum binderi) was

124 collected. This was performed a few successive times until a clear filtrate was obtained at the

125 end of the treatment. The defatted seaweed (44.5 g) was then dried in a vacuum oven
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126 (Vacucell MMM, Munich, Germany) at 50°C for 24 h. Extraction of fucoidan from

127 Sargassum binderi was performed using a 2% CaCl2 solution at 85°C with automated

128 mechanical stirring (200 rpm) for 24 h. This step was repeated six times to ensure complete

129 fucoidan extraction. The extracts were combined and added with 10%

130 hexadecyltrimethylammonium bromide solution (in excess) to precipitate the fucoidan. The

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131 precipitate formed was centrifuged (Eppendorf Centrifuge 5810R, 3000 g, 10 min) and rinsed

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132 with water to remove excess hexadecyltrimethylammonium bromide before being stirred in

133 20% ethanolic sodium iodide (Fisher Scientific) solution (5 × 100 ml) for 4-5 days, rinsed

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134 with ethanol to remove excess sodium iodide, and dissolved in water. The fucoidan solution

135 was then dialysed against distilled water for 4-5 days using a Visking tube with a molecular

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weight cut-off (MWCO) of 2000 Da, before being lyophilised to obtain fucoidan as a sodium
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137 salt in solid form (Fsar) (Bilan et al., 2004).
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139 2.5 Identification and purity of fucoidan

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140 2.5.1 High performance anion exchange chromatography (HPAEC)

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141 Fucoidan identification and purity determination were achieved using high

142 performance anion exchange chromatography (HPAEC) with a Waters system

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143 (Massachusetts, USA) equipped with an auto injector as performed by Rioux et al. (2007),
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144 with modifications to the temperature and flow rate parameters. The anion exchange column
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145 used was Phenomenex Rezex RPM monosaccharide, 100 × 7.8 mm, with a guard column

146 (Phenomenex 50 × 7.8 mm). The isolated fucoidan (Fsar) and commercial food grade fucoidan

147 (Fysk) were prepared at 5 mg/ml concentrations, and 20 µl was injected into the HPAEC

148 system. The elution was conducted using deionised water at a flow rate of 0.2 ml/min and a

149 temperature of 85°C. The eluent was detected using an evaporative light scattering detector

150 (Polymer Laboratories, PL-ELS 1000) with an evaporator temperature of 120°C, a nebuliser
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151 temperature of 90°C, a transfer line temperature of 30°C and a gas flow rate of 1.2 slm. The

152 Waters system works with Breeze software. A fucoidan standard (Fsig) was used for the

153 identification, while the determination of purity was achieved using Fysk (92% purity) to

154 provide a standard curve. A factor of 92% was used in the calculation of purity of Fsar.

155

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156 2.5.2 Attenuated total reflectance Fourier transform infra-red (ATR-FTIR) spectroscopy

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157 Attenuated total reflectance Fourier transform infra-red (ATR-FTIR) spectroscopy

158 was performed using a Perkin Elmer Precisely, Spectrum 400, FT-IR/FT-NIR spectrometer

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159 (Massachusetts, USA) fitted with a Spectrum Universal ATR Accessory. A total of 4 scans

160 were performed on the samples with a scanning range of 4000 - 650 cm-1 and a resolution of

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4 cm-1 (Rodriguez-Jasso et al., 2011; Souza et al., 2012). The vibrational spectra were
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162 collected and analysed using Essential FTIR [Link] software.
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163

164 2.6 Determination of antioxidant capacity

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165 2.6.1 Total Phenolic Content (TPC)

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166 The TPC of fucoidan was measured using the Folin-Ciocalteu method as described by

167 Chew et al. (2008). Samples of Fsar and Fysk were prepared at 2 mg/ml. The samples (0.3 ml)
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168 were mixed with 1.5 ml Folin-Ciocalteu reagent and 1.2 ml sodium carbonate solution (7.5%
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169 w/v). The reaction mixtures were incubated in the dark for 90 min before measuring the
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170 absorbance at 765 nm in a 96-well plate (200 µl) using a BioTek Epoch microplate

171 spectrophotometer. A standard curve of gallic acid with a concentration range of 5-50 ppm

172 was prepared, and the TPC was expressed as gallic acid equivalents per 100 g sample

173 (GAE/100 g). The TPC of Fsar and Fysk were compared with those of BHA, BHT and ascorbic

174 acid at the same concentration. An n=3 replication was conducted on the total phenolic

175 content.
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176

177 2.6.2 Free radical scavenging assay (DPPH)

178 Free scavenging activity was measured based on the scavenging ability of stable 1,1-

179 diphenyl-2-picrylhydrazyl (DPPH) radicals by Fsar and Fysk as described by Chew et al.

180 (2008). A series of Fsar and Fysk concentrations (0.05-0.5% w/v) were prepared, and 1 ml of

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181 each Fsar and Fysk concentration was mixed with 2.9 ml methanolic 0.15 mM DPPH. After

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182 incubation of the reaction mixture in the dark for 30 min, the absorbance was measured at

183 517 nm in a 96-well plate (200 µl) using a BioTek Epoch microplate spectrophotometer. The

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184 DPPH tests were performed in triplicate. The IC50 of Fsar and Fysk were calculated and

185 compared with that of BHA, BHT and ascorbic acid using the formula below:

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187 % Free radical scavenging activity = [(A1 – A2)/A1] × 100 Eq. (1)
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188

189 where A1 is the absorbance of the DPPH blank and A2 is the absorbance of the sample with
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190 DPPH.
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191

192 The DPPH radical scavenging were then expressed in terms of ascorbic acid
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193 equivalent antioxidant capacity (AEAC), which was calculated based on the equivalent
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194 antioxidant capacity of ascorbic acid with 100 g of sample as follows:

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196 AEAC (mg AA/100 g) = [IC50(AA) / IC50(sample)] × 100,000 Eq. (2)

197 where AA is ascorbic acid and IC50 is the inhibition concentration at 50%.

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199

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201 2.6.3 Reducing power assay

202 The reducing power assay was performed according to Kumar et al. (2008). Both the

203 Fsar and Fysk, which were prepared at 2 mg/ml, were mixed with phosphate buffer (2.5 ml, 0.1

204 M, pH 6.6) and potassium ferricyanide [K3Fe(CN)6] (2.5 ml, 1% w/v). The solutions were

205 then incubated at 50°C for 20 min. Trichloroacetic acid (2.5 ml, 10%) was added to the

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206 solutions, and the mixtures were centrifuged at 2000 g for 10 min (Eppendorf Centrifuge

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207 5810R). A portion of the samples’ upper layers (2.5 ml) were mixed with 2.5 ml distilled

208 water and 0.5 ml 0.1% (w/v) FeCl3 and were left at room temperature for 30 min before

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209 measuring the absorbance at 700 nm in a 96-well plate (200 µl) using a BioTek Epoch

210 microplate spectrophotometer. The reducing power assay was performed in triplicate. The

211
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results were expressed as gallic acid equivalents (mg GAE/100 g) and compared with BHA,
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212 BHT and ascorbic acid (2 mg/ml).
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213

214 2.6.4 Superoxide anion (SOA) scavenging activity

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215 The superoxide anion scavenging activity was determined by measuring the inhibition
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216 of pyrogallol auto-oxidation (Heo et al., 2005; Marklund & Marklund, 1974). Both Fsar and

217 Fysk were prepared at 2 mg/ml. The samples (0.3 ml) were mixed with 2.6 ml phosphate
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218 buffer (50 mM, pH 8.24) and 90 µl of 3 mM pyrogallol solution (dissolved in 10 mM HCl).
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219 The absorbance, which reflects the inhibition rate of pyrogallol auto-oxidation, was measured
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220 at 325 nm in a 96-well plate (200 µl) using a BioTek Epoch microplate spectrophotometer in

221 which the absorbance was recorded every 1 min interval for 10 min. A replication of n=3 was

222 performed in the superoxide anion scavenging activity test. The percentages of superoxide

223 anion scavenging activities of the samples were compared with those of BHA, BHT and

224 ascorbic acid (2 mg/ml) and were calculated using the following equation:

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226 Scavenging rate (%) = [1-(A2-A1)/A0] × 100 Eq. (3)

227 where A1 is the absorbance of the sample at zero min, A2 is the absorbance of the sample at

228 the tenth min, and A0 is the autoxidation rate of pyrogallol for the blank (the change of

229 absorbance in the blank from zero min to ten min).

230

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231 2.6.5 Hydroxyl radical scavenging activity (·OH)

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232 The hydroxyl radical scavenging activity (·OH) was determined according to Zhao et

233 al. (2012). Both Fsar and Fysk were prepared at 2 mg/ml. The hydroxyl radical was generated

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234 using the Fenton reaction, where 0.5 ml of 9 mM ferrous sulphate (FeSO4) solution was

235 added to 1.0 ml of 8.8 mM hydrogen peroxide (H2O2) (35% purity, HmbG Chemicals)

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solution. This mixture was then added to the Fsar and Fysk samples prepared earlier, before
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237 adding 0.2 ml of 9 mM salicylic acid solution. Another set of reaction solutions was prepared
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238 as above without the addition of salicylic acid. The mixtures were allowed to stand at 37°C

239 for 1 h before the absorbance was measured at 510 nm in a 96-well plate (200 µl) using the
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240 BioTek Epoch microplate spectrophotometer. The hydroxyl radical scavenging activities of
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241 the samples were compared with those of BHA, BHT and ascorbic acid at the same

242 concentration (2 mg/ml). The hydroxyl radical scavenging activity was determined in
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243 triplicate, and the activity was calculated as follows:

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244
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245 Hydroxyl radical scavenging rate (%) = [1-{(A1-A2)/A0}] × 100 Eq. (4)

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247 where A0 is the absorbance of the blank (without sample), A1 is the absorbance of the

248 reaction containing the sample and salicylic acid, and A2 is the absorbance of the reaction

249 containing the sample but without salicylic acid.

250
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251 2.7 Statistical analysis

252 Analysis was performed in triplicate (n=3), except for the HPAEC and FTIR analyses,

253 which were done once. Data were obtained as the mean and standard deviation and analysed

254 using one-way ANOVA followed by Duncan’s multiple range test (DMRT) using SAS

255 version 6.12 for Windows. The difference in mean values was considered significant when

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256 p<0.05.

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257

258 3. Results and discussion

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259 3.1 Screening and isolation of fucoidan content

260 The screening of the fucoidan content was performed using a colorimetric method in

261
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which two different wavelengths, 396 nm (λ396) and 427 nm (λ427), were used to determine
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262 the fucose content via the specific colour reactions of fucose with L-cysteine and sulphuric
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263 acid. The difference in absorbance between the two wavelengths (∆λ396-λ427) differentiates

264 fucose from other hexoses present, as the absorbance of fucose has a maximum at λ396 but is
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265 near zero at λ427. At the same time, the absorbance of all other hexoses is the same at both
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266 wavelengths of λ396 and λ427 (Dische & Shettles, 1948; Usov et al., 2001). This test acted as

267 the pre-confirmation of fucoidan presence in the seaweeds.

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268 As shown in Table 1, the brown seaweeds, i.e. Sargassum binderi (6.16 ± 0.08%) and
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269 Padina sp. (2.06 ± 0.23%), showed the presence of fucoidan at significantly higher (p<0.05)
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270 amounts compared to those in the red seaweeds. The low readings from the red seaweeds

271 might not be from fucoidan, as explained by Usov et al. (2001), where the fucose detected in

272 red seaweeds was actually the product of a colour reaction from other 6-deoxyhexoses found

273 in seaweed. However, as a rule, brown seaweeds do not contain these compounds, and the

274 colour reactions with other sugars are excluded by the colour measurement at the two

275 wavelengths (Usov et al., 2001).

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276 As discussed by Wijesinghe and Jeon (2012), it was found in this pre-confirmation

277 test that brown seaweeds are the main source of fucoidan. Sulphated polysaccharides are

278 among the most abundant and broadly studied polysaccharides from non-animal origin, and

279 brown seaweeds are recognised as a major source of sulphated polysaccharides (fucoidan)

280 with various biological activities. Due to the high fucoidan content in Sargassum binderi in

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281 the former work, further isolation work was performed on this particular species for the

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282 determination of its antioxidant capacity. In the extraction and isolation of fucoidan, a total of

283 3.34 g of fucoidan (Fsar) was successfully isolated from 44.5 g of Sargassum binderi, giving a

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284 7.5% yield.

285 Studies have been conducted on the fucoidan content of various species of brown

286
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seaweed collected in different seasons and locations. These studies showed that the fucoidan
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287 content varies with the species, season and location. A notable study by Usov et al. (2001),
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288 who studied the fucoidan content of 17 species of brown seaweed collected in July 1990 from

289 Avancha Bay, Kamchatka, Russia, showed that the fucoidan content ranged from 0.4%
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290 (Desmarestia intermedia P. et R., Laminaria dentigera Kjellm, and Arthrothamnus bifidus P.
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291 et R.) to 20.4% (Saudersella simplex (Saund.) Kylin).

292 Another study conducted by Zvyagintseva et al. (2003) showed variations in the
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293 fucoidan content from brown seaweeds of different species, seasons and locations, where
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294 samples were collected from the Troitsa Bay (Laminaria cicorioides) and the Rifovaya Bay
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295 (Laminaria japonica), Japan and from a few islands in the Sea of Okhotsk, Russia (Fucus

296 evanescens). These samples were collected in different seasons (from August 1996 to August

297 1999). It was shown that the fucoidan content in terms of dry weight basis varied from 1.1%

298 to 12.0% depending on the species, season and location. A more recent study by Rioux et al.

299 (2009), who collected brown seaweed (Saccharina longicruris) from two locations, Percé

300 and L'Anse-à-Beaufils, both in Québec, Canada, between May 2005 to June 2006, showed
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301 that the fucoidan content from this species in different seasons and locations varied from

302 1.6% to 4.5%.

303 All these studies concluded that brown seaweeds contain fucoidan and that their

304 content varies according to the species, season and location. Therefore, this justifies the study

305 of fucoidan extracted from Malaysian brown seaweed, Sargassum binderi, as there are

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306 differences in fucoidan from different sources. The fucoidan yield of 7.5% (Fsar) is

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307 acceptable, as it is within the range of fucoidan content from the various studies discussed

308 above.

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309

310 3.2 Identification of fucoidan

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3.2.1 High performance anion exchange chromatography (HPAEC)
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312 An elution using high performance anion exchange chromatography (HPAEC) was
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313 performed on the Fsar and Fysk, which were compared to the fucoidan standard (Fsig). This

314 acted as a primary confirmation test. The chromatograms of the HPAEC (Figure 1) show that
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315 the retention time of Fsig was 10.537 min, while Fsar and Fysk had retention times of 10.343
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316 min and 10.632 min respectively. The Breeze system matched all three chromatogram peaks

317 as the same compound; i.e. fucoidan. Therefore, the primary confirmation of fucoidan was
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318 successfully achieved. The purity of Fsar was successfully determined at 89.63%,using Fysk to

plot the standard curve with an R2=0.9927.

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319
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320

321 3.2.2 Attenuated total reflectance Fourier transform infra-red (ATR-FTIR) spectroscopy

322 A secondary confirmation test on Fsar was then carried out using ATR-FTIR

323 spectroscopy. The ATR-FTIR spectra of both Fsar and Fysk are shown in Figure 2. This

324 analysis was conducted to determine whether Fsar and Fysk have similar infra-red absorption

325 properties to those identified in published data. Both the Fsar and Fysk spectra showed similar
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326 signals, with two bands in the 4000-2000 cm-1 region and numerous signals with similar

327 wavenumbers within the 2000-800 cm-1 region. Common to all polysaccharides and seaweed

328 polysaccharides, two bands appeared in the 4000-2000 cm-1 region of the ATR-FTIR spectra.

329 There was a broad band centred at 3378 cm-1 (Fsar) and 3383 cm-1 (Fysk), which was assigned

330 to hydrogen bonded O-H symmetrical and asymmetrical stretching vibrations (Gomez-

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331 ordonez & Ruperez, 2011; Coimbra et al., 1998). These peaks were also caused by the

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332 presence of moisture in the sample, as H2O contains O-H bonds that give a stretching

333 vibration signal (Silverstein & Webster, 1998). There was another weak signal at 2944 cm-1

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334 (Fsar) and 2941 cm-1 (Fysk) due to C-H stretching vibrations (Gomez-ordonez & Ruperez,

335 2011; Coimbra et al., 1998). Additionally, the medium to strong IR absorption bands (1200-

336
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970 cm-1) are mainly due to C-C and C-O stretching in pyranoid rings and to C-O-C
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337 stretching in glycosidic bonds. Intense absorption in this spectral region is common for all
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338 polysaccharides (Synytsya et al., 2010).

339 The typical absorption bands of fucoidan are the bands that indicate the presence of
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340 sulphate (SO4) and methyl (CH3) groups, as fucoidan is a sulphated polysaccharide and
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341 contains mainly fucose, a monosaccharide that has a methyl group attached to the C5 position

342 (Ale et al., 2011; Li et al., 2008). The signals at 1420 cm-1 (Fsar) and 1414 cm-1 (Fysk) were
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343 attributed to the presence of the asymmetrical bending vibration of CH3, while the signals at

1369 cm-1 (Fsar) and 1367 cm-1 (Fysk) were assigned to the symmetrical bending vibration of
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344
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345 CH3 (Silverstein & Webster, 1998; Kim et al., 2010).

346 There was a strong signal at 1025 cm-1 (both Fsar and Fysk) and a shoulder signal at

347 1076 cm-1 (Fsar) and 1075 cm-1 (Fysk), which were assigned to the stretching vibrations of

348 sulphoxides (S=O). The strong signals for sulphoxides indicate that there were significant

349 amounts of sulphate groups in the fucoidan (Silverstein & Webster, 1998; Marais & Joseleau,

350 2001). A rather sharp signal at 963 cm-1 (Fsar) and 958 cm-1 (Fysk) indicates the presence of
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351 the asymmetrical stretching vibration of C-O-S bonds, while the signals at 822 cm-1 (Fsar) and

352 837-849 cm-1 (Fysk) were assigned to the symmetrical stretching vibrations of C-O-S bonds

353 (Synytsya et al., 2010). At the same time, the absorption at 822 cm-1 (Fsar) indicates the

354 presence of sulphate groups at the equatorial C2 and C3 positions, while the absorption at

355 837-849 cm-1 (Fysk) was attributed to the sulphate groups at the axial C4 position (Patankar et

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356 al., 1993).

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357 Therefore, as mentioned above, the presence of sulphate and methyl groups as well as

358 of C-O-S bonds, which are typical in fucoidan ATR-FTIR spectra, enabled us to identify the

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359 polysaccharide extracted from Sargassum binderi (Fsar) as fucoidan. The signals at 1619 cm-1

360 (Fsar) and 1616 cm-1 (Fysk) were due to the bending vibrations of HOH, which indicate the

361
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presence of moisture in the samples (Silverstein & Webster, 1998). Another peak at 1732 cm-
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1
362 appeared in the Fysk spectra, and this peak was due to the C=O stretching vibrations of
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363 carboxylic esters (Gomez-ordonez & Ruperez, 2011; Silverstein & Webster, 1998). This peak

364 was not as evident in the Fsar spectra because it was masked by the stronger HOH bending
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365 vibration.
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366

367 3.3 Antioxidant assays

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368 In this research, the free radical scavenging activity (DPPH) assay examines the
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369 primary antioxidant potential, while the superoxide anion (SOA) scavenging activity and
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370 hydroxyl radical (·OH) scavenging activity assays examine the secondary antioxidant

371 potential. The reducing power assay, on the other hand, examines both primary and

372 secondary antioxidant capacity. There are generally two types of antioxidants, namely,

373 primary and secondary antioxidants, as mentioned above. Primary antioxidants, also referred

374 to as chain-breaking antioxidants, function as free radical acceptors/scavengers and delay or

375 inhibit the initiation step or interrupt the propagation step of auto-oxidation. Secondary
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376 antioxidants, on the other hand, are classified as preventive antioxidants, which slow the rates

377 of oxidation reactions through various mechanisms. Secondary antioxidants act as chelators

378 for prooxidants (metal ions), provide H to primary antioxidants, decompose hydroperoxide to

379 nonradical species, deactivate singlet oxygen, absorb ultraviolet radiation or act as oxygen

380 scavengers. Therefore, the main difference between primary and secondary antioxidants is

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381 that secondary antioxidants do not convert free radicals into stable molecules (Wanasundara

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382 & Shahidi, 2005).

383

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384 3.3.1 Total phenolic content

385 According to Table 2, the total phenolic content (TPC) of Fsar (3.69 ± 0.15 mg

386
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GAE/100 g) was not significantly (p>0.05) different from that of Fysk (3.64 ± 0.23 mg
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387 GAE/100 g), but both Fsar and Fysk had significantly (p<0.05) lower TPC than BHA, BHT and
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388 ascorbic acid.

389 This was expected because fucoidan is not a phenolic compound but rather an
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390 aliphatic polysaccharide with monosaccharides, namely, fucose, galactose, xylose, glucose,
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391 mannose and glucuronic acid, as its building blocks. In contrast, BHA, BHT and ascorbic

392 acid have aromatic (phenolic) structures and therefore gave high TPC readings (Bilan et al.
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393 2004).
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394
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395 3.3.2 Free radical scavenging activity (DPPH)

396 Antioxidants that have free radical scavenging activity can reduce free radicals, such

397 as DPPH radicals. DPPH is a stable radical that possesses a nitrogen free radical with a

398 maximum absorbance at 517 nm (Kumar et al., 2008; Yangthong et al., 2009). Due to the

399 simplicity and convenience of this assay, it is used widely in free-radical scavenging activity

400 assessments (Williams et al., 1995).

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401 Based on the results shown in Table 2, it was found that the IC50 of Fsar was not

402 significantly (p>0.05) different from that of Fysk, but both Fsar and Fysk possessed significantly

403 (p<0.05) higher IC50 values than BHA, BHT and ascorbic acid. A lower IC50 is favourable

404 because it indicates that a lower concentration is needed for 50% inhibition of the DPPH free

405 radical. The same observation was made for the ascorbic acid equivalent antioxidant capacity

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406 (AEAC), whereby both Fsar and Fysk were not significantly (p>0.05) different. AEAC is the

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407 equivalent of the antioxidant capacity of ascorbic acid (in mg) in 100 g of sample. This shows

408 that the extracted fucoidan (Fsar) has a lower free-radical scavenging activity than that of

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409 synthetic antioxidants. Therefore, it was prudent to carry out further studies on its secondary

410 antioxidant capacity.

411
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412 3.3.3 Reducing power
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413 In terms of reducing power, it was found that both Fsar and Fysk did not show

414 significant (p>0.05) differences, but again, both were significantly (p<0.05) lower than that
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415 of BHA, BHT and ascorbic acid (Table 2). This shows that Fsar and Fysk exhibited low
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416 reducing power.

417 Antioxidants with reducing power are those that can act as electron donors and can
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418 reduce the oxidised intermediates of lipid peroxidation processes, allowing them to act as
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419 primary and secondary antioxidants (Chanda & Dave, 2009). Such antioxidants react with

potassium ferricyanide (Fe3+) to form potassium ferrocyanide (Fe2+), which then reacts with
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420

421 ferric chloride (Fe3+) to form ferrous complexes (Fe2+) that have a maximum absorbance at

422 700 nm (Ferreira et al., 2007; Jayanthi & Lalitha, 2011). Therefore, based on the DPPH and

423 reducing power test results, both Fsar and Fysk have low primary antioxidant activity.

424

425
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426 3.3.4 Superoxide anion scavenging activity

427 Superoxide anion radicals are formed from cellular oxidation reactions in organisms,

428 including in humans. Although it is a relatively weak oxidant, it decomposes to produce

429 stronger oxidative species, such as hydrogen peroxide and hydroxyl radicals, through

430 dismutation and other types of reactions. It is also the source of the free radicals formed in

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431 vivo. SOA radicals and its derivatives are cell-damaging, causing damage to DNA and cell

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432 membranes. Therefore, it is of great important to scavenge SOA radicals (Sarikurkcu et al.,

433 2010; Yangthong et al., 2009).

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434 In this assay, the results in Table 2 show that the superoxide anion (SOA) scavenging

435 activity of Fsar (26.78 ± 1.90% inhibition) was significantly (p>0.05) higher compared to that

436
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of Fysk (20.57 ± 1.48% inhibition), BHA (6.41 ± 0.69% inhibition) and BHT (24.41 ± 1.27%
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437 inhibition). Ascorbic acid had the significantly highest superoxide anion scavenging activity
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438 among all the samples. The superoxide anion scavenging activity was measured using the

439 pyrogallol auto-oxidation system, and the results were expressed as the inhibition rate of
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440 superoxide production. The significantly (p>0.05) higher SOA scavenging activity of Fsar
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441 compared to that of Fysk, BHA and BHT demonstrates the secondary antioxidant potential of

442 Fsar.
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443
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444 3.3.5 Hydroxyl radical scavenging activity (·OH)

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445 The hydroxyl radical is the most reactive free radical and can be formed from

446 superoxide anions and hydrogen peroxides in the presence of metal ions, such as copper and

447 iron. Hydroxyl radicals can cause damage to nearly all types of biomolecules, including

448 proteins, DNA, polyunsaturated fatty acids, and nucleic acids (Aruoma, 1998). These

449 damages accelerate the aging process and cause cancer and several diseases (Zhu et al.,
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450 2006). Therefore, the removal or scavenging of hydroxyl radicals in the human body is one of

451 the most effective defences by the body.

452 As shown in Table 2, Fsar (60.95 ± 0.69%) has significantly higher (p<0.05) hydroxyl

453 radical scavenging activity compared to Fysk (40.22 ± 2.68%) and BHT (35.71 ± 2.62%). It

454 was also found that the hydroxyl radical scavenging activity of Fsar was not significantly

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455 different (p>0.05) compared to that of BHA (63.75 ± 1.57%). Ascorbic acid had the highest

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456 (p<0.05) hydroxyl radical scavenging activity compared to the rest of the samples.

457 The tests of both superoxide anion and hydroxyl radical scavenging activities showed

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458 that Fsar demonstrates effective secondary antioxidant capacity. Therefore, the fucoidan

459 extracted from Malaysian brown seaweed, Sargassum binderi, has the potential to be a source

460 of natural secondary antioxidants.

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461
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462 3.3.6 Comparison of antioxidant activities with those in previous works

463 In this research, it was clearly established that Fsar showed lower primary but higher
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464 secondary antioxidant activity when compared to synthetic antioxidants. The relatively low
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465 primary antioxidant activity indicates that Fsar does not scavenge free radicals effectively. In

466 contrast, the high secondary antioxidant activity of Fsar showed that it can scavenge or quench
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467 pro-oxidants, such as oxygen. Interestingly, Wanasundara and Shahidi (2005) reported that
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468 secondary antioxidants also function as H-donors to primary antioxidants, allowing

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469 secondary antioxidants to regenerate primary antioxidants. This is a form of synergistic effect

470 between primary and secondary antioxidants.

471 Various studies have been conducted on the antioxidant activity of the fucoidan

472 extracted from different species of brown seaweed. Similar antioxidant activities were

473 observed in all these previous works, with the fucoidan demonstrating antioxidant activity at

474 different levels depending on the type of antioxidant assay. According to Wang et al. (2010)
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475 and Wang et al. (2008), who studied the antioxidant activity of the fucoidan extracted from

476 Laminaria japonica (cultured in Shazikou, Qingdao, China – August 2007 and March 2005),

477 the IC50 of the DPPH scavenging activity of the fucoidan was 3.7 mg/ml, which is higher than

478 that of the current work, at 2.01 mg/ml (a lower IC50 is more favourable). The study also

479 reported that the extracted fucoidan displayed high superoxide anion and hydroxyl radical

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480 scavenging activities and that the superoxide anion scavenging activity was higher than that

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481 of BHA.

482 Another study by Souza et al. (2007) demonstrated that the fucoidan extracted from

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483 Fucus vesiculosus and Padina gymnospora showed significant superoxide anion and

484 hydroxyl radical scavenging activities in terms of its IC50 values. The current work showed

485
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that both the superoxide anion and hydroxyl radical scavenging activities of Fsar are
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486 significantly higher than (p<0.05) or do not differ significantly (p>0.0.5) from those of BHA
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487 and BHT. In a review paper by Li et al. (2008), the authors stated that fucoidan from various

488 sources showed significant inhibition of superoxide anion and hydroxyl radical scavenging
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489 activities, while a lower DPPH radical scavenging activity was observed.
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490 A study by Kuda et al. (2005) showed a similar trend in antioxidant activity as that

491 observed in the current work. The study used the brown seaweed Scytosiphon lomentaria
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492 harvested in Wajima, Ishikawa, Japan, in March 2003. It was discovered that the reducing
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493 power and the DPPH scavenging activity of the crude fucoidan were relatively low compared
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494 to that of ascorbic acid. While no particular dilutions were given, the concentrations of crude

495 fucoidan used in the study were approximately (based on the values from the graphs) 0.3

496 mg/ml to 5 mg/ml (a stock solution of 5 mg/ml was prepared). The IC50 of the DPPH

497 scavenging activity was not determined in this concentration range, which shows that the

498 fucoidan from the current work (Fsar) has better DPPH scavenging activity (IC50 = 2.01
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499 mg/ml). In the same study, it was reported that crude fucoidan showed superoxide anion

500 scavenging activity.

501 Therefore, the Fsar from the current work has potential applications as a natural

502 antioxidant, especially in terms of secondary antioxidants. The Fsar gave significantly higher

503 (p<0.05) values or no significant difference (p>0.05) compared with Fysk in all antioxidant

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504 assays performed in the current work. It was also found that the antioxidant capacity of Fsar is

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505 similar or superior to that found in previous studies.

506

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507 4. Conclusions

508 Among the screened samples, Sargassum binderi had the highest fucoidan content.

509
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The polysaccharide extracted from Sargassum binderi (Fsar) (yield 7.5%) was identified as
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510 fucoidan (purity 89.63%) through HPAEC and ATR-FTIR spectroscopy methods and
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511 exhibited antioxidant capacity. It was found that Fsar showed lower primary antioxidant

512 activity but significantly higher secondary antioxidant activity, namely, superoxide anion and
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513 hydroxyl radical scavenging activity, compared to that of the synthetic antioxidants (BHA
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514 and BHT). The antioxidant properties of Fsar were comparable to those of commercial, food-

515 grade fucoidan (Fysk), as both the superoxide anion and hydroxyl radical scavenging activities
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516 of Fsar were significantly higher (p<0.05) than that of Fysk, and all the other antioxidant assays
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517 showed no significant difference (p>0.05) between Fsar and Fysk. In addition, Fsar was found to
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518 have a superior or at least similar antioxidant capacity compared to that observed in previous

519 studies. Therefore, Malaysian brown seaweed (Sargassum binderi) is a good source of

520 fucoidan that exhibits antioxidant activity and has the potential to be commercialised as a

521 natural antioxidant bioingredient.

522

523
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524 Acknowledgements

525 This research was funded by STGL-007-2010 and OUP-2012-128 grants and the

526 MyBrain15 (MyPhD) Scholarship by the Ministry of Higher Education, Malaysia. The

527 authors would like to thank SIRIM Bhd, Malaysia for supplying the seaweed samples, Yaizu

528 Suisankagaku Industry Co., Ltd., Yaizu City, Japan for providing commercial food-grade

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529 fucoidan and the School of Chemical Sciences and Food Technology, Faculty of Science and

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530 Technology, Universiti Kebangsaan Malaysia, which provided all the facilities necessary for

531 this research.

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532

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695 41, 1296–1302.

696

697 Zvyagintseva, T.N., Shevchenko, N.M., Chizhov, A.O., Krupnova, T.N., Sundukova, E.V., &

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698 Isakov, V.V. (2003). Water-soluble polysaccharides of some far-eastern brown

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699 seaweeds. Distribution, structure, and their dependence on the developmental

700 conditions. Journal of Experimental Marine Biology and Ecology, 294, 1– 13.

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702 Zvyagintseva, T.N., Shevchenko, N.M., Popivnich, I.B., Isakov, V.V., Scobun, A.S.,

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704 water-soluble polysaccharides from brown seaweeds. Carbohydrate Research, 322,
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705 32-39.

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Fig. 1 HPAEC chromatograms of (a) fucoidan standard (Fsig), (b) fucoidan isolated from

Sargassum binderi (Fsar) and (c) commercial food grade fucoidan (Fysk)

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I : O-H stretching vibrations

II : C-H stretching vibrations
III : Presence of moisture
IV : CH3 asymmetrical bending vibrations
V : CH3 symmetrical bending vibrations

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VI : S=O stretching vibrations
VII : C-O-S asymmetrical stretching vibrations

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VIII : C-O-S symmetrical stretching vibrations

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Fig. 2 Attenuated total reflectance - Fourier transform infra-red (ATR-FTIR) spectra of (a)

fucoidan isolated from Sargassum binderi (Fsar) and (b) commercial food grade fucoidan (Fysk)

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Table 1 Fucoidan content of seaweed samples (n=3)

Sample Fucoidan content (%)

Brown seaweed

Sargassum binderi 6.16 ± 0.08a

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Padina sp. 2.06 ± 0.23b

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Red seaweed

Gracilaria sp. 0.27 ± 0.08c

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Eucheuma spinosum 0.33 ± 0.06c

Eucheuma cottonii 0.36 ± 0.10c

a-d

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Table 2 Comparison of antioxidant activities of the fucoidan extracted from Sargassum binderi (Fsar), commercial food-grade fucoidan
(Fysk), BHA, BHT and ascorbic acid (n=3)

DPPH

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TPC Reducing power SOA inhibition ·OH scavenging
Sample IC50 AEAC
(mg GAE/100g) (mg GAE/100g) (%) (%)
(mg/ml) (mg AA/100g)

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Fsar 3.69 ± 0.15c 2.01 ± 0.29a 17.64 ± 2.82a 0.60 ± 0.08d 26.78 ± 1.90b 60.95 ± 0.69c

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Fysk 3.64 ± 0.23c 1.87 ± 0.08a 18.71 ± 0.87a 0.26 ± 0.01d 20.57 ± 1.48d 40.22 ± 2.68d

172.94 ± 13.26b (5.96 ± 0.01)×10-4 b 63.61 ± 0.27a 6.41 ± 0.69e 63.75 ± 1.57c

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BHT 101.43 ± 18.60b (9.59 ± 0.05)×10-4 b - 32.41 ± 0.78c 24.41 ± 1.27c 35.71 ± 2.62e

Ascorbic acid 451.75 ± 79.05a (3.49 ± 0.19)×10-4 b - 42.26 ± 0.27b 98.85 ± 0.25a 99.97 ± 0.15a

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Highlights

• Fucoidan was highest in Sargassum binderi among 5 seaweed species screened.

• HPLC and FTIR results confirmed that the compound extracted was fucoidan.
• Purity of the extracted fucoidan was 89.63%.
• Fucoidan isolated from S. binderi has significant secondary antioxidant potential.

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• Both isolated and commercial food grade fucoidan has comparable antioxidant capacity.

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