Food Chemistry 136 (2013) 1055–1062

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Food Chemistry

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Fucoxanthin content and antioxidant properties of Undaria pinnatifida

Adah Fung a, Nazimah Hamid a, Jun Lu a,b,c,⇑a School of Applied Sciences, Faculty of Health and Environment Sciences, Auckland University of Technology, Auckland, New Zealandb School of Interprofessional Health Studies, Faculty of Health and Environment Sciences, Auckland University of Technology, Auckland, New Zealandc Institute of Biomedical Technologies, Auckland University of Technology, Auckland, New Zealand

a r t i c l e i n f o a b s t r a c t

Article history:Received 31 May 2012Received in revised form 3 August 2012Accepted 3 September 2012Available online 17 September 2012

Keywords:FucoxanthinUndariaAntioxidantNew ZealandSeasonal variation

0308-8146/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.foodchem.2012.09.024

⇑ Corresponding author at: School of Applied ScieEnvironment Sciences, Auckland University of TechnZealand. Tel.: +64 9 9219999x7381; fax: +64 9 92191

E-mail address: jun.lu@aut.ac.nz (J. Lu).

This study investigated the fucoxanthin content of New Zealand (NZ) Undaria pinnatifida harvested fromtwo locations in the Marlborough Sounds, New Zealand across its growing season. Fucoxanthin contentand antioxidant properties of processed New Zealand U. pinnatifida and commercial wakame from Japanand Korea were further compared. Results showed that U. pinnatifida harvested from Port Underwood hadhigher fucoxanthin content in the blade compared to Pelorus Sound. The sporophyll also contained a sig-nificant amount of fucoxanthin throughout the harvest season, although lower than in the blade. Twoantioxidant measurement methods, DPPH and CUPRAC, were utilised to measure antioxidant activities.Processed NZ U. pinnatifida had a lower fucoxanthin content and antioxidant activity than freeze-driedUndaria. Fucoxanthin content and antioxidant activities of NZ processed U. pinnatifida were not signifi-cantly different from other commercial samples from Japan and Korea. In conclusion, U. pinnatifida inNew Zealand has a great potential to be a food and nutraceutical resource.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

In answer to societies demand for a better lifestyle and in-creased longevity, consumers have developed an increasing inter-est towards consuming nutraceuticals and functional foods thatare rich in natural bioactive compounds. Antioxidants are amongthe well-known bioactive compounds required by the human bodyin order to stay healthy. Antioxidants inhibit further oxidationreactions that produce free radicals, which cause cell damage ordeath, and consequently induce different kinds of serious chronicdiseases, including cancer and atherosclerosis. Seaweeds are richin polysaccharides, vitamins, minerals, and bioactive substanceslike polyphenols, proteins, lipids and carotenoid that possess anti-oxidant, antibacterial, antiviral and other beneficial functions. Re-cently, marine products are of considerable interests in the foodand pharmaceutical industries as a potential source of antioxidants(Ngo, Wijesekara, Vo, Van Ta, & Kim, 2010).

The strong antioxidant activities in seaweeds are present aschemical protection mechanisms. Macroalgae are frequently ex-posed to a combination of strong light and high oxygen concen-trations that lead to the formation of reactive oxygen species(ROS) and other strong oxidising agents. However damage to

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nces, Faculty of Health andology, Auckland 1142, New75.

the structural components (polyunsaturated fatty acids) or anyserious photodynamic damage of the algae seldom occurs. In or-der to survive these harsh marine environments, seaweeds havedeveloped strong protective antioxidative defence systems thatconsist of an array of antioxidative compounds that may ormay not work synergistically to limit oxidation. Several studieson different seaweed species have shown that brown seaweedshave a higher antioxidant capacity than red or green seaweed(Jiménez-Escrig, Jiménez-Jiménez, Pulido, & Saura-Calixto, 2001;Matanjun, Mohamed, Mustapha, Muhammad, & Ming, 2008; Pra-bhasankar et al., 2009). Phlorotannins are the only group of tan-nins present in brown seaweed. These extracts have shownprotective effects against hydrogen peroxide-induced cell dam-age by acting as free radical scavengers (Tierney, Croft, & Hayes,2010), reducing agents and metal chelators (Ngo et al., 2010;Tierney et al., 2010). On the contrary, fucoxanthin is the domi-nant carotenoid in brown seaweeds. Although less attentionhas been paid to the physiological effects of carotenoid in sea-weeds, fucoxanthin has recently attracted much attention dueto its strong antioxidant properties that showed significantanti-cancer, anti-obesity and anti-inflammatory effects (Miyash-ita & Hosokawa, 2008). Fucoxanthin in its pure form is vulnera-ble to oxidation. Nonetheless it was found to be fairly stable inthe presence of co-existing antioxidants such as polyphenol.Fucoxanthin identified in the dried form of algae stored at ambi-ent temperature (Miyashita & Hosokawa, 2008) was present inlow amounts indicating that the process of drying could have

Fig. 1. Figure of Undaria pinnatifida.

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decomposed fucoxanthin. The content of fucoxanthin was re-ported to vary significantly with season and life cycle of the al-gae, peaking between the winter and spring (mature phase ofsporophyte) and lowest during summer (senescence phase) (Ter-asaki et al., 2009).

Undaria pinnatifida is a type of brown seaweed that normallygrows in the seas of Japan. Its structure can be divided into theblade (lamina), midrib, sporophyll and root-like formations (hap-tera) as shown in Fig. 1. Its Japanese name is wakame, which is alsothe name of the processed U. pinnatifida that is used as food. In1987, this seaweed was accidently introduced into New Zealandwaters by shipping. It was classified as an invasive species andan unwanted organism in New Zealand under Section 164c of theBiosecurity Act 1993. However in late 2010, a new policy wasimplemented that allowed farming and harvesting of U. pinnatifidafor commercial use. Although U. pinnatifida has been in New Zea-land for more than 20 years, no studies have investigated its poten-tial commercial use as a food and as a source of antioxidant.

Due to the presence of different components in the crude extractsof seaweed, it was relatively difficult to measure each antioxidantcomponent separately. Therefore, two assay methods were em-ployed in this study to evaluate the total antioxidant activity of U.pinnatifida samples. The DPPH�(2,2-diphenyl-1-picrylhydrazyl or1,1-diphenyl-2-picrylhydrazyl) scavenging assay utilises a stablenitrogen centred free radical. DPPH are effectively scavenged byantioxidants through the donation of hydrogen to form the reducedDPPH–H (Brand-Williams, Cuvelier, & Berset, 1995). This methodhas been widely used in the antioxidant measurement of brown sea-weeds (Airanthi, Hosokawa, & Miyashita, 2011; Chew, Lim, Omar, &Khoo, 2008; Jiménez-Escrig, et al., 2001; Karawita, Siriwardhana, &L., 2005). The CUPRAC (cupric ion reducing antioxidant capacity) as-say utilises a chromogenic redox reagent, bis(neocuproine) copper(II) chelate that can be reduced to a Cu(I)-chelate by a reducing anti-oxidant (Apak et al., 2007). This method is a variant of the ferricreducing antioxidant power (FRAP) assay. It has been widely usedto measure the antioxidant capacity in fruits (Guclu, Altun, Ozyurek,Karademir, & Apak, 2006), vegetables (Koksal & Gulcin, 2008) andplants (Apak et al., 2007; Celik et al., 2008). In this study, DPPH scav-enging activities are indicative of the hydrogen donating ability ofthe seaweed extract to scavenge free radicals, whereas the CUPRACmethod is a measure of the reducing capacity of the seaweed extract.

This study sought to examine fucoxanthin content in freeze-dried New Zealand U. pinnatifida across its growing season. Fuco-xanthin content and antioxidant properties in processed New Zea-land U. pinnatifida and commercial wakame from Japan and Koreawere further compared.

2. Materials and method

2.1. Materials and sample preparation

Methanol, hexane and chloroform were purchased from Ther-mofisher (Auckland, New Zealand) and were of HPLC grade. 2,2-di-phenyl-1-picrylhydrazyl (DPPH), fucoxanthin and canthaxanthinstandards were purchased from Sigma–Aldrich (St. Louis, MO,USA). Japanese and Korean wakame (U. pinnatifida) commercialproducts were purchased from a Korean Supermarket in Auckland,New Zealand.

U. pinnatifida seaweeds were harvested on a monthly basis fromJune 2011 to November 2011. The seaweeds were obtained fromtwo farms located in Port Underwood; farm PE327 (41� 200

53.0500 and 174� 070 20.9600) and farm 106 (41� 190 37.7400 and174� 080 57.5400), and two farms from the Pelorus Sound; farm122 (41� 060 30.8900 and 173� 540 58.0500) and farm 353 (41� 010

56.9500 and 173� 560 12.5500) located in the Marlborough Sounds,New Zealand (Fig. 2). Samples collected from each farm werewashed several times with seawater on the boat to remove foreignmatters and sand, and were then dried with paper towels. Bladeand sporophyll parts were separated and kept in individual bags.These samples were then frozen overnight and air-freighted toVitaco Limited, Avondale, Auckland, to be lyophilised in bulk with-in 48 h of frozen storage. All freeze-dried U. pinnatifida sampleswere pulverized (Breville CG2B Coffee ‘n’ Spice Grinder) and sieved(600 lm sieve) to obtain a fine powder. Each sample was stored inindividual 200 ml PET bottles and kept in the dark at room temper-ature until use.

2.2. Fucoxanthin extraction

All fucoxanthin extraction was carried out away from directsunlight to reduce the possibility of oxidation by sunlight. Thefreeze dried sample (100 mg) was mixed with methanol (15 ml)and stirred using a magnetic bar for an hour at room temperature.Solids were removed using a Whatman No.1 filter paper (Thermo-fisher, New Zealand). Hexane (15 ml) and water (15 ml) wereadded to the methanol extract and vortexed for 1 min and left topartition into two distinct layers using a separation funnel. Theupper phase was discarded and the lower phase was vortexed for1 min with 10 ml of chloroform to extract fucoxanthin. The mix-ture was centrifuged (Sorvall RC5C instruments, Fiberlite F21–8x50y rotor, Thermofisher USA) at 17,000�g for 15 min at 4 �C.The aqueous phase was removed, leaving the organic layerundisturbed.

The organic phase was then dried at 30 �C using a rotary evap-orator. Methanol (5 ml) was added to the dried extract. The result-ing solution was transferred into a glass vial, flushed with argongas and stored at �80 �C until further use. Seven U. pinnatifida sam-ples (comprising both blade and sporophyll) from each farm wereselected for further analysis.

2.2.1. HPLC analysisFucoxanthin was determined using a HPLC method (Terasaki

et al., 2009). The HPLC system consisted of a LC-20AT pump system(Shimadzu), a UV–Vis SPD-20A (Shimadzu) absorbance detectorand online analysis software (LC solution version 1.25). Fucoxan-thin was separated on a Luna 5 lm C18 (2) 250 � 4.6 mm column(Phenomenex, Auckland, New Zealand). The mobile phase usedwas 100% methanol with a flow rate of 1 ml/min and the sampleinjection volume was 20 ll. The detection wavelength was set at450 nm.

A 5 point calibration curve was constructed with a detectionrange from 0.0078 to 0.125 lg/ml using pure fucoxanthin standard

Fig. 2. Map of the Marlborough Sounds showing mussel farm locations where U. pinnatifida samples were collected.

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dissolved in methanol. Canthaxanthin standard (0.03125 lg/ml fi-nal concentration) dissolved in acetone was used as an internalstandard. Each fucoxanthin standard curve set was injected induplicates before and after the injections of all U. pinnatifida ex-tracts. The lower and upper limits of detection were 0.00236 lg/ml and 0.379 lg/ml, respectively.

2.3. Processing of New Zealand U. pinnatifida

Cut wakame is a popular dried wakame product that can beconsumed once rehydrated. Since one of the aims of this studywas to investigate the antioxidant potential of the wakame productmade from New Zealand U. pinnatifida, the U. pinnatifida sampleswere processed using a similar method used to produce commer-cial wakame (Kantono, 2011). Fresh New Zealand U. pinnatifidafrom Farm 327 Port Underwood in the Marlborough Sounds washarvested in July, August and September 2011. Samples wererinsed, placed into plastic bags on the boat and transported tothe AUT laboratory via airfreight. Samples were washed repeatedlywith tap water to remove sand, defective leaves and other foreignobjects. Salt (300 g) was mixed with 1 l of water and heated to80 �C using a hot plate. Samples were blanched at 80 �C for1 min, cooled quickly in a beaker filled with ice water for anotherminute, and then cured (3:10 w/w ratio of salt to sample) for 48 h.The cured samples were then briefly rinsed and placed into plasticbags that were pierced. Heavy weights were placed on the bags for48 h to further remove water. After pressing, the samples weredried for 24 h at 60 �C using an oven. Dried U. pinnatifida wasplaced into zip-lock bags and stored in the dark at room tempera-ture until further analysis.

2.4. Extraction of antioxidants

All antioxidant extractions of commercial Japanese (Wako Sho-kai and Riken), and Korean wakame products (Ottogi and ChungJung Won), and the laboratory-processed New Zealand wakamesamples were carried out away from direct sunlight to reducethe possibility of oxidation. Methanol (15 ml) was added to the

sample (300 mg) and stirred for an hour at room temperature. Sol-ids were removed using a Whatman No.1 filter paper (Thermofish-er, New Zealand). The methanol extract was then dried using arotary evaporator at 30 �C. Methanol (20 ml) was added to thedried extract. The resulting solution was transferred into a glassvial, flushed with argon gas and stored at �80 �C until furtheruse. Analysis of each sample was repeated five times.

2.5. Analysis of antioxidant activity

2.5.1. DPPH radical scavenging activityDPPH radical scavenging activity was measured using the meth-

od of Duan, Zhang, Li and Wang (2006). Two millilitre of samplewas mixed with 2 ml of 0.16 mM DPPH in methanol and incubatedat room temperature for 30 min in the dark. After incubation, theabsorbance was measured at 517 nm. The reading on the spectro-photometer (Ultraspec 2100 pro UV/VIS spectrophotometer, Amer-sham Pharmacia Biotech.) was zeroed using methanol as theblank.The scavenging activity was calculated as follows:

Scavenging ð%Þ ¼ 1� Abssample � Abssample blank

Abscontrol

� �� 100

where Abssample was the absorbance of the sample with treatment,Abssample blank was the absorbance of sample with 2 ml of methanol,and Abscontrol was the absorbance of 2 ml of methanol with thetreatment.

2.5.2. CUPRACThe CUPRAC reagent was prepared according to the method de-

scribed by Apak, Guclu, Ozyurek, and Karademir (2004). Copper (II)chloride solution (10�2M) was prepared by weighing 426.2 mg ofCuCl2�2H2O and dissolving it in 250 ml distilled water. Ammoniumacetate (NH4Ac) buffer at pH 7.0 was prepared by dissolving19.27 g of NH4Ac into 250 ml of distilled water. Neocuproine (Nc)solution (7.5 � 10�3 M) was prepared by dissolving 3.9 mg of Ncin 96% ethanol that was further diluted to 25 ml with 96% ethanol.

One ml of Cu (II), Nc and NH4Ac buffer solutions were addedinto a test tube. Sample (1 ml) and distilled water (0.1 ml) were

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then added to the initial mixture to make up a final volume of4.1 ml. The test tubes were manually shaken for a few secondsand left in the dark for 30 min. After incubation, the absorbancewas measured at 450 nm against a reagent blank. The reading onthe spectrophotometer was zeroed using distilled water as theblank. The absorbance of each sample was recorded.

2.6. Statistical analysis

One-way analysis of variance (ANOVA) was carried out to testfor differences in the fucoxanthin content of freeze-dried U. pin-natifida samples between months, farms and locations using Mini-tab� (Version 16). Results of all tests were considered significant atP < 0.05. The association between fucoxanthin and antioxidantactivities of New Zealand U. pinnatifida was evaluated using Pear-sons correlation (Minitab� Version 16). One-way ANOVA was fur-ther used to analyse differences between fucoxanthin content andantioxidant activities of processed New Zealand U. pinnatifida andcommercial wakame products. Where there were significant differ-ences, a post-hoc Tukey’s test was employed to examine wherethat effect occurred. All data analysed using ANOVA met theassumptions of equal variance and homogeneity as determinedusing Minitab� (Version 16).

3. Results and discussion

3.1. Fucoxanthin content in U. pinnatifida

The HPLC chromatogram of fucoxanthin extracted from theblade and sporophyll of New Zealand U. pinnatifida is shown inFig. 3. Fucoxanthin was detected using the UV–VIS spectrum at450 nm. The peak corresponding to all-trans-fucoxanthin was de-tected at a retention time of 3.6 min. The two peaks ascribed asthe 13-cis and 130-cis isomers of fucoxanthin were detected atretention times of 4.3 and 4.5 min, respectively. The peak detectedat a retention time of 8.3 min was the internal standard, cantha-

Fig. 3. HPLC chromatograms of fucoxanthin in (a) blade and (b) sporophyll extracts of Nefucoxanthin; Peak 4, unidentified peak; Peak 5, canthaxanthin as the internal standard.

xanthin. An unidentified peak was detected at a retention time of6.2 min in both blade and sporophyll. However the unidentifiedpeak was not purified for further identification as this was not anobjective of the research carried out.

3.2. Fucoxanthin content

3.2.1. BladeA significant increase (p < 0.001) in fucoxanthin content was ob-

served in the blade of seaweeds obtained from farms 327 and 106in Port Underwood starting from June and reached a maximum inJuly 2011 (Fig. 4a). This was followed by a significant decrease(p < 0.001) in fucoxanthin content in August that remained con-stant in September before a rapid decrease (p < 0.001) in Novem-ber. Collection of U. pinnatifida from the Pelorus Sound (farms122 and 353), was only carried out in August, September, Octoberand November as the seaweeds only started growing in August2011 (Fig. 4c). Fucoxanthin content in the blade from farm 122was significantly higher compared with farm 353 in August(p = 0.001) and September (p = 0.007). Samples from Farm 122 inAugust, September and October had significantly higher(p < 0.001) fucoxanthin content than in November. However, therewas no significant difference between the monthly changes infucoxanthin content from farm 353. Differences observed in thefucoxanthin content between farms from the same location couldbe due to the proximity of the farms from each other. Farms 327and 106 were located approximately 2 km apart, whereas farms122 and 353 were located approximately 10 km away (Fig. 2).The close proximity of farms 327 and 106 in Port Underwood madeit likely that these farms were exposed to similar environmentalfactors such as nutrients, sunlight, and temperature that have beenreported to influence the growth of seaweeds and their pigmentcontents (Campbell, Bite, & Burridge, 1999; Dean & Hurd, 2007;Terasaki et al., 2009).

The change in fucoxanthin content in the blade of Undaria ob-tained from Port Underwood over its growing season was similar

w Zealand U. pinnatifida Peak 1, all-trans-fucoxanthin; Peaks 2 and 3, cis-isomers of

Fig. 4. Fucoxanthin content (mg/g) of U. pinnatifida collected from June to November 2011 froms the Marlborough Sounds, New Zealand. (a) blade from Port Underwood,farms 327 and 106; (b) sporophyll from Port Underwood, farms 327 and 106; (c) blade from Pelorus Sound, farms 122 and 353; (d) sporophyll from Pelorus Sound, farms 122and 353. All the values are mean ± standard error of seven samples (period when seaweeds were not collected are left blank). Significant differences (p < 0.05) with monthsfor blade or sporophyll from a farm are indicated by different superscript lower letters. Significant differences (p < 0.05) with months between farms within a location; PortUnderwood (farm 327 and farm 106) and Pelorus Sound (farm 122 and farm 353) for each body part (blade or sporophyll) are indicated by different superscript capital letters.

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to U. pinnatifida harvested from Carey’s Bay at the Otago Harbour,New Zealand from May to November 1996 (Dean & Hurd, 2007).The authors reported that the total pigment content, includingfucoxanthin, increased steadily from May, reaching a maximumin July, decreased (p < 0.001) in August, and remained constant un-til a rapid decrease (p < 0.001) in November 1996. Hence, U. pin-natifida collected from the Marlborough Sounds had a similargrowth pattern to the U. pinnatifida in the Otago Harbour.

3.2.2. SporophyllFucoxanthin content in the sporophyll (Fig. 4b and d) was sig-

nificantly less (p < 0.001) than blade (Fig. 4a and c) for all monthsand all four farms investigated. This may be because sporophyllsare responsible for reproduction. Accessory pigments such as fuco-xanthin are more abundant in the blade in order to harvest light forgrowth. Nevertheless, sporophyll contained significant amounts offucoxanthin across all months and farms (�20–50% of fucoxanthinin the blade) that can be further extracted for commercial use. Assporophyll is often discarded when U. pinnatifida is harvested asa food source, extraction of fucoxanthin from the sporophyll willnot only reduce waste but also turn it to a valuable source ofhealth/nutraceutical products.

3.3. Comparison of monthly variations in the fucoxanthin content ofblade and sporophyll between two locations

Fucoxanthin content in the blade and sporophyll of U. pinnatifi-da collected from August and November from two locations (PortUnderwood and Pelorus Sound) were compared because thesewere the only months where samples were collected in all four

farms (Fig. 4). Port Underwood was found to be a better locationto harvest U. pinnatifida as fucoxanthin content was high in bothblade and sporophyll, especially in August 2011. The differencesin fucoxanthin content between seaweeds harvested from PortUnderwood and Pelorus Sound could be due to differences in thegrowing environment. Environmental factors are crucial in deter-mining the growth and pigment content of seaweed (Barr & Rees,2003; Campbell et al., 1999; Dean & Hurd, 2007). In our study, U.pinnatifida in the Pelorus Sound grew later than those in PortUnderwood. This limitation resulted in the comparison of samplesobtained from Port Underwood and Pelorus Sound in the months ofAugust and November, 2011 only. As Pelorus Sound was mainlysurrounded by land and located further away from the open seacompared with Port Underwood (Fig. 2), the later growth of U. pin-natifida in the Pelorus Sound may be due to a higher freshwater in-put from the surrounding land that washed into the sea.Freshwater was found to slow down the growth of seaweed andcould even kill seaweed (Anderson, 2007; Trioba, 2007). The useof freshwater pumped from the Torrens River into saline ‘‘WestLakes’’ in South Australia was reported to have successfully con-trolled and eradicated the Caulerpa taxifolia (Chlorophyta) seaweed(Anderson, 2007).

3.4. Differences in fucoxanthin content between processed and freezedried New Zealand U. pinnatifida

In our study, there was an average 51.8% reduction in the fuco-xanthin content of processed New Zealand samples compared withthe freeze dried samples (Table 1). The processing of U. pinnatifidasamples into a wakame food product involved blanching the sea-

Table 1Fucoxanthin content (mg.g�1), DPPH (2,2-diphenyl-1-picrylhydrazyl free radical) radical scavenging assay (%) and CUPRAC assay (absorbance) of fresh and processed NewZealand U. pinnatifida.

Fucoxanthin content DPPH radical scavenging activity CUPRAC assay

Type Processed Fresh Processed Fresh Processed Fresh

MonthJuly 2.08 ± 0.04Aa 4.96 ± 0.45Ba 32.09 ± 3.03Aa 87.18 ± 4.04Ba 0.93 ± 0.07Ba 1.41 ± 0.11Aa

August 1.77 ± 0.02Aa 4.28 ± 0.33Bab 35.34 ± 3.51Aa 91.29 ± 3.37Ba 0.78 ± 0.02Ba 1.30 ± 0.08Aa

September 2.04 ± 0.21Aa 3.32 ± 0.51Bb 39.64 ± 2.50Aa 85.01 ± 4.65Ba 0.81 ± 0.02Ba 1.20 ± 0.16Ab

All the values are mean ± standard error of seven samples. Significant differences (p < 0.05) with months for processed or fresh U. pinnatifida are indicated by differentsuperscript lower letters. Significant differences (p < 0.05) between processed and fresh U. pinnatifida with months are indicated by different superscript capital letters.

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weed in 80 �C water for 1 min, curing with salt for 48 h to removewater, and finally oven drying for 24 h at 60 �C (see Section 2.3).Thermal processing of the seaweed may thus be the main factorresponsible for reduction in fucoxanthin in the processed samples.This is supported by Mise, Ueda, and Yasumoto, (2011) who re-ported that freeze dried seaweed Cladosiohon okamuranus (Pha-eophyta) gave the highest fucoxanthin content compared to hot-wind dried and vacuum dried samples. Fucoxanthin content wasreduced by 51% in the samples dried with hot-wind at 60 �C com-pared to the freeze dried sample The process of drying seaweed athigh temperature decomposed fucoxanthin and even if fucoxan-thin was able to withstand the drying process, it was susceptibleto oxidation when exposed to higher temperatures (Mise, 2011).In addition, the fact that U. pinnatifida was cured with salts for48 h may also reduce fucoxanthin content. The solute rich environ-ment results in osmotic pressure that draws water out of the sea-weed and retards the growth of microorganism. Curing wasreported to deactivate enzymes in seaweed to prolong its productshelf life (Nisizawa, Noda, Kikuchi, & Watanabe, 1987; Watanabe &Nisizawa, 1984).

3.5. Differences in fucoxanthin content between New Zealandprocessed and commercial U. pinnatifida products

Fucoxanthin content of New Zealand processed U. pinnatifidaharvested in July from farm 327 in Port Underwood, was comparedwith commercial Japanese and Korean wakame products. Fucoxan-thin content of New Zealand processed U. pinnatifida was not sig-nificantly different to the commercial dried Korean products(Ottogi and Chung Jung Won), but was significantly lower(p < 0.001) than the Japanese products (Wako Shokai and Riken)(Table 2). This could be due to variation in the fucoxanthin contentof the fresh seaweed with locations and months of harvest (Airan-thi et al., 2011). Furthermore, differences in heat processingparameters may also be responsible for the differences in the fuco-xanthin content of processed New Zealand U. pinnatifida comparedto commercial Japanese products (Wako Shokai and Riken). For in-stance, hot wind drying of seaweed was found to decrease fuco-

Table 2Variation in the fucoxanthin content (mg.g�1), DPPH (2,2-diphenyl-1-picrylhydrazylfree radical) radical scavenging assay (%) and CUPRAC assay (absorbance) in NewZealand (NZ) processed and commercial U. pinnatifida from Japan and Korea.

Products Fucoxanthin content DPPH (%) CUPRAC assay

NZ 2.08 ± 0.04a 32.09 ± 3.03a 0.92 ± 0.07b

Ottogi 2.00 ± 0.07a 31.35 ± 1.88a 1.20 ± 0.05a

Chung Jung Won 1.91 ± 0.05a 31.49 ± 2.51a 1.23 ± 0.02a

Wako Shokai 2.89 ± 0.17b 26.02 ± 3.93b 1.19 ± 0.08a

Riken 2.81 ± 0.02b 29.53 ± 3.48b 1.09 ± 0.02b

All the values are mean ± standard error of seven samples for fucoxanthin content.Significant differences (p < 0.05) between months for each column are indicated bydifferent superscript lower letters.

xanthin content more compared to vacuum drying (Mise, 2011).Increasing the temperature in hot wind and vacuum drying canalso increase the decomposition of fucoxanthin in seaweed (Mise,2011). In Japan, cut wakame was reported to be traditionally pre-pared by drying in a flow through dryer that pass through sievesto sort the different sized pieces (Nisizawa et al., 1987).

3.6. Antioxidant activity

3.6.1. DPPH scavenging and CUPRAC reducing activities of NewZealand processed and fresh U. pinnatifida

In this study, both the DPPH radical scavenging and CUPRAC as-says showed a significant reduction (59% and 35%, respectively) inthe antioxidant activities of processed U. pinnatifida compared tothe fresh samples (Table 1). It has been reported that processedseaweeds generally contained less antioxidant activity than freshseaweeds (Jiménez-Escrig et al., 2001; Yan, Chuda, Suzuki, & Nag-ata, 1999).

It has been determined that the antioxidant effect of seaweedwas mainly due to the antioxidant activity of phenolic compounds(Airanthi et al., 2011; Jiménez-Escrig et al., 2001). In this study, arelationship between the fucoxanthin content and antioxidantactivities was evident, with DPPH showing a stronger correlationbetween fucoxanthin (r2 = 0.879) than the CUPRAC (r2 = 0.752) as-say. Hence, the reduction of antioxidant activity of processed sea-weed compared to fresh samples in our study could be due to theloss of polyphenol and fucoxanthin during processing. The DPPH�scavenging assay has been extensively employed to study the rad-ical scavenging capacity of seaweed. The major active compoundsin brown seaweed extracts detected with the DPPH�scavenging as-say have been reported to be polyphenol and fucoxanthin (Airanthiet al., 2011; Ganesan, Kumar, & Bhaskar, 2008). The radical scav-enging capacity of seaweed was reported to be mostly related totheir phenolic hydroxyl group (Jiménez-Escrig et al., 2001). Simi-larly, the correlation of Folin total phenolic contents of herbal teaswith CUPRAC antioxidant capacities gave a linear correlation coef-ficient of 0.966 implying that the results of CUPRAC correlated wellwith the total phenolics content of herbal infusions (Celik et al.,2008). This is the first reported study using the CUPRAC assay toevaluate antioxidant activity of seaweed. A reasonable correlationwas found between fucoxanthin and the CUPRAC assay (r2 = 0.752).Further investigations are required to assess the involvement ofother antioxidant activities measured using the CUPRAC assayapart from polyphenol and fucoxanthin in seaweed extracts.

Like fucoxanthin, drying also decreased the phenolic content byinactivating the enzymes, polyphenol oxidases (Jiménez-Escriget al., 2001; Lim & Murtijaya, 2007). Jimenez-Escrig et al. (2001) re-ported that the total phenolic content of processed Fucus vesiculo-sus (class Phaephyceae) decreased by 98% and DPPH� scavengingactivity decreased by 96% compared to the fresh material (Jimé-nez-Escrig et al., 2001). However, for New Zealand processed U.pinnatifida, the decrease in the DPPH� scavenging activity was not

A. Fung et al. / Food Chemistry 136 (2013) 1055–1062 1061

as drastic, with an average of 52% reduction. This could be due to U.pinnatifida being processed differently and/or contained a higheramount of other strong antioxidants that were not susceptible toloss during processing. Overall, the reduction of antioxidant activ-ities in New Zealand processed U. pinnatifida compared to freezedried samples as determined by the DPPH radical scavenging andCUPRAC assays could be due to the loss of polyphenol and fucoxan-thin during processing.

3.6.2. Comparison of DPPH�scavenging and CUPRAC reducing activitiesbetween New Zealand processed and commercial U. pinnatifidaproducts

DPPH scavenging activities in NZ, Ottogi and Chung Jung Wonwakame samples were significantly higher (p < 0.001) comparedto Wako Shokai and Riken (Table 2). However, the CUPRAC assayshowed that the reducing activity in Ottogi, Chung Jung Won,and Wako Shokai samples were significantly higher (p < 0.001)compared to NZ processed U. pinnatifida and Riken.

Phlorotannins, the largest group of polyphenols have been re-ported to be the main antioxidant in brown seaweeds (Shibata,Ishimaru, Kawaguchi, Yoshikawa, & Hama, 2008; Yuan & Walsh,2006). They exist in a variety of structures due to the different de-gree of polymerisation (Ngo et al., 2010). In this study, differencesin the DPPH radical scavenging activity and CUPRAC assays couldbe partly due to structural variations in the phenolic compoundsand the selectivity of the antioxidant reacting to the assays. TheCUPRAC reagent was reported to be more selective (Apak et al.,2004). Simple sugars and citric acid, which are not true antioxi-dants are not oxidised with the CUPRAC reagent that was alsocapable of measuring thiol-type antioxidants such as gluthione.CUPRAC can also simultaneously measure hydrophilic and lipoph-illic antioxidants, whereas the DPPH radical scavenging assay wassolvent dependant (Huang, Ou, & Prior, 2005; Prior, Wu, & Schaich,2005).

The variation in antioxidant activities observed between NewZealand processed U. pinnatifida and commercial wakame productsin both assays may also be due to the involvement of antioxidantsother than polyphenols. Fucoxanthin and tocopherol from brownseaweed have been demonstrated to have DPPH radical scavengingactivity (Le Tutour et al., 1998; Sachindra et al., 2007; Yan et al.,1999). Levels of the tocopherols in seaweed tissue were found tovary with season. The differences in months and harvest locationsof U. pinnatifida might further account for the differences in theantioxidant compounds of the New Zealand and commercial wak-ame products.

4. Conclusions

Fucoxanthin content in New Zealand Undaria exhibited locationvariation and also varied depending on the life cycle of the sea-weeds indicating the possible biological significance of this pig-ment in seaweeds. U. pinnatifida harvested from Port Underwoodhad higher fucoxanthin content in the blade than Pelorus Sound.U. pinnatifida harvested in Port Underwood during winter con-tained a significant amount of fucoxanthin. Fucoxanthin was alsofound in the sporophyll of U. pinnatifida throughout the growingseason, even though its level was significantly lower than that inthe blade. However as sporophyll is often discarded as it is not con-sumed as food, it can instead be a useful bioresource for fucoxan-thin. Processed New Zealand U. pinnatifida had comparable amountof fucoxanthin, as well as antioxidant activity, compared to com-mercial wakame products analysed. However as processing re-duced the amount of fucoxanthin, an effective stabilisationmethod will be necessary to apply fucoxanthin in nutraceuticalsand formulation of functional foods. The pathogen free aquatic

environment and relative absence of inorganic toxins of New Zea-land waters may further give New Zealand U. pinnatifida a compet-itive edge in the production of high quality fucoxanthin. In thisstudy, the uniqueness of the antioxidant activities of New ZealandU. pinnatifida was their significant radical scavenging activities andreducing capacities compared to commercial fucoxanthin thatexhibited either a stronger scavenging activity or reducing capacitybut not both. This could be due to the different growing environ-ment of New Zealand U. pinnatifida that may produce uniquechemical compounds possessing antioxidant activities that reactedin both antioxidant assays.

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