Australian Journal of Basic and Applied Sciences, 4(10): 4580-4584, 2010
ISSN 1991-8178
Stability Studies of Fucoxanthin From Sargassum Binderi
1Siew-Ling Hii, 2Pooi-Yi Choong, 1Kwan-Kit Woo, 2Ching-Lee Wong 1Department of Chemical Engineering, 2Department of Science, Faculty of Engineering and Science, Universiti Tunku Abdul Rahman, Jalan Genting Kelang, Setapak,
53300 Kuala Lumpur, Malaysia.
Abstract:F ucoxanthin is an important carotenoid component available in all brown algae. In this study, Sargassum binderi was used as the source of fucoxanthin and the main purpose of the study was to evaluate the stability of fucoxanthin extract against the different storage conditions. Stabilities of the extracted fucoxanthin were tested with different pH and with supplementation of antioxidant
(i.e., ascorbic acid) in light and dark condition over a storage period of 4 weeks. Colour changes of
the fucoxanthin extract were monitored using colour software CIE LAB system. The results revealed that fucoxanthin pigment exhibited the greatest stability when stored in dark condition. The pigments were more stable at alkaline pH region as compared to neutral and acidic condition. In dark condition, the addition of ascorbic acid greatly delayed fucoxanthin degradation and concentration ascorbic acid at 1.0% w/v displayed greatest pigment retention. In conclusion, fucoxanthin pigments were sensitive to light exposure and acidic pH condition and could be stabilised by higher concentration of ascorbic acid.
Key words: F ucoxanthin, S. binderi, Stability, Colour software
INTRODUCTION
reactive materials studiesPigments, as a form of nutrient found in seaweeds, have important nutraceutical properties, including antioxidant, besides providing anti-obesity, anticancer and anti-inflammation effect (Miyashita and Hosokawa, 2007; Maeda et al., 2008). Apart from that, seaweeds have been used for many medical purposes in several traditional medical systems, including nutritional support, immune stimulation and body detoxification (Maeda et al., 2008).
F ucoxanthin, a yellowish brown pigment belonging to the xanthophylls of carotenoid pigments is found
abundantly in brown algae. The pigment fucoxanthin has attracted much attention as a thermogenic agent, besides contains several other functional components that could benefit an individual with obesity (Maeda et al., 2005). A study proved that the anti-obesity effect of fucoxanthin, through protein and gene expressions mitochondrial uncoupling protein 1 (UCP1) in white adipose tissue (WAT). The results revealed that fucoxanthin up-regulated the expression of UCP1 in WAT and hence contributed to reduction of WAT weight. It is an ideal therapy of obesity as most of the fat is stored in WAT (Maeda et al., 2005, Maeda et al., 2008). Efficiency of fucoxanthin in reducing the viability of prostate cancer cells was also reported (Kotake-Nara et al., 2001). Another beneficial effect of fucoxanthin is the chemoprevention of cancer in which fucoxanthin possesses the inhibitory effect on cancer cells by inhibiting the growth of human neuroblastoma cells and intestinal carcinogenesis (Mori et al., 2004). In poultry breeding and aquaculture, fucoxanthin were excellent feed additive as it increases the yellow color of egg yolk when fed to poultry and the metabolic fate of fucoxanthin in laying hens (Strand et al., 1998).
As a type of carotenoids, fucoxanthin are highly susceptible to degradation by external agents, such as heat, low pH, and light exposure, promoting changes of colour due to several conjugated double bonds (Mercadante, 2008). The degradation process would lead to the rearrangement or formation of
degradation compounds such as cis-isomers which are thermodynamically less stable and hence resulted in different colours properties and in some cases, volatile compounds (Mercadante, 2008).
The present study was undertaken to extract the pigment of fucoxanthin from Sargassum binderi and subsequently, to evaluate the stability of fucoxanthin that subjected to different treatment methods and storage conditions.
Corresponding Author:Siew-Ling Hii, Department of Chemical Engineering,
E-mail: hiisl@; phone: +603-41079802; fax: +603-41079803
MATERIALS AND METHODS
Specimen collection:
The seaweeds (Sargassum binderi) were collected from Cape Rachado, Port Dickson, Malaysia. The freshly collected were rinsed successively with distilled water to remove sand, epiphytes and saline ions. The seaweeds were left for air-dried in the laboratory for few days. The dried seaweeds were ground into powder form. Extraction of fucoxanthin:
The powderised seaweeds were treated with acetone solution for 2 h in the dark condition. Following that, absolute methanol was added and the mixture was stirred for 15 min in room temperature. The supernatant were then vacuum-filtered to remove the seaweed solid particles. The resulting acetone filtrate was concentrated at 40 C, at 556 mbar in a rotary evaporator. The concentrated extract was stored at -20 C for further analysis on its stability.
Stability studies of fucoxanthin:
For pH stability test, the initial pH of fucoxanthin pigment extract were adjusted to desired pH (pH 3, pH 5, pH 7 and pH 9) by using 1 M HCl or 1 M NaOH, prior to storage. Sample with pH 7 was used as control. For the investigation of antioxidant effect on fucoxanthin stability, L-ascorbic acid were added to the aliquots containing fucoxanthin pigment extract to achieve the desired concentration (% w/v) of 0.1%, 0.5% and 1.0%. Control was also prepared with no addition of L-ascorbic acid. All samples were prepared in triplicates.
A total of two sets of triplicate were prepared as the stability of pigment was studied for both light and dark condition. The samples were then subjected to 4 consecutive weeks of observation. Spectrophotometric Analysis of Fucoxanthin Concentration:
Concentrations of fucoxanthin were measured at the l
of 425 nm by using a single-beam visible
max
spectrophotometer (Genesys 20, Thermo Scientific).
Color characteristics including lightness (L*) and chroma value (colour intensity) were measured using a double-beam UV-Visible spectrophotometer (Lambda 35 UV/Vis Spectrum, Perkin-Elmer) equipped with UVWinLab Version 2.85.04 and a colour software, Wincol Version 2.05 Perkin-Elmer Advanced Spectroscopy Colour Application Software.
Statistical analysis:
The data obtained in this study were statistically analysed  by using the SPSS 11.5 software (SPSS 11.5 for Windows® software, SPSS Incorporation). Statistical analysis was performed to determine and compare the relationship between different sample treatments in different storage conditions by one-way analysis of variance (ANOVA) and Post Hoc Duncan test. Significance of difference was defined at p < 0.05.
RESULTS AND DISCUSSION
Effect of Antioxidant Additives on Fucoxanthin Stability:
As shown in F igure 1, despite the addition of ascorbic acid, fucoxanthin pigment degraded drastically immediate after one week of storage, in all samples which were exposed to the artificial light source. In contrast, with the addition of ascorbic acid, fucoxanthin pigment exhibited the greatest stability with approximately 50% of pigment retained when stored in dark condition (Table 1). F rom statistical analysis, samples without the addition of ascorbic acid exhibited the least pigment retention while with the addition of 1% w/v of ascorbic acid, the fucoxanthin pigment possessed greater stability as compared to others. This phenomenon suggested that fucoxanthin is at its most stable state with higher concentration of ascorbic acid supplementation.
Ascorbic acid is a powerful antioxidant owing to the ability to donate a hydrogen atom and form a relatively stable ascorbyl free radical (Weber et al., 1996). Being a  scavenger of reactive oxygen and nitrogen oxide species, ascorbic acid showed effectiveness in combating superoxide radical ion, hydrogen peroxide, the hydroxyl radical and singlet oxygen (Yan et al., 1999). F rom Table 1, the degradation rate of pigment was slower with the aid of ascorbic acid and ascorbic acid at 1% w/v prov
ed to be a good treatment for preservation of the colour.  In addition, antioxidant such as ascorbic acid is a molecule capable of slowing and preventing the oxidation of other molecules. Fucoxanthin is a major carotenoid in brown algae and it has the property of easily oxidized (Sinninghe Damsté & Koopmans, 1997). Hence, addition of ascorbic acid might be favourable and thus contributing to the stability of fucoxanthin.
Fi g. 1: Stability of fucoxanthin treated with ascorbic acid and exposed to light
Effect of pH:
The stabilities of fucoxanthin in different pH condition (pH 3, pH 5, pH 7 and pH 9) were studied over a storage period of four weeks in light and dark condition. The extracted fucoxanthin exhibited greater stability in dark condition when pH of solution was adjusted to pH 9 (Table 2). According to Hui (2006), carotenoids are relatively stable at alkaline pH and it was suggested that the pigment could retain pigment better in alkaline condition as compared to acidic condition.  Furthermore, acid is known to initiate degradation of carotenoids in food and beverages by a largely unknown mechanism (Chen et al., 1995).  Factors such as acids could also promote trans–cis isomerization reactions and lead to the degradation of pigment (Gliemmo et al., 2009).
The stability of fucoxanthin was the least in the pH 3 which suggested that fucoxanthin was relatively unstable in acidic condition (Table 2). Carotenoids have been found to be protonated by nitric acid and moderately strong acids such as trichloroacetic acid and trifluoroacetic acid, except for carotenoids bearing the hydroxyl group (Mortensen and Skibsted, 2000). However, the protonation site varies where carotenoids containing carbonyl groups are preferentially protonated on this group and not on a
carbon atom of the conjugated system. F or carotenoids containing hydroxy groups, there was only a slight alteration of the carotenoid recativity (Mortensen and Skibsted, 2000). Thus, this contributed to the higher stability of carotenoids with carbonyl groups compared to that of other xanthophylls and carotenes.
According to a study conducted by Chen et al. (1995), at high pH (pH 10 and pH 8), the rate of degradation of carotene and xanthophylls contents of lucerne juice was significantly slower than at acidic pH (pH 6.5 and pH 5.4). Hence, it could possible be verified that acidity increased degradation of carotenoids, with the presence of other degradation mechanisms of carotenoids such as heat and light.
Effect of light:
Carotenoids are sensitive towards light and hence easily degraded by light (Mortensen & Skibsted, 2000). In the present study, accelerated degradation of pigment was highly observed in the presence of light under the various condition of pH and antioxidant addition (Table 1 and Table 2). F actor such as light promotes trans-cis isomerisation reactions and eventually explained the fading colour of the pigment (Gliemmo et al., 2009). Besides that, it might also be accompanied by the formation of new co
mpounds due to oxidation which would give rise to loss of color (Arita et al., 2005) and in some cases, formation of aroma compounds as well (Mortensen and Skibsted, 2000). According to Arita et al. (2005), light is a potential reason for degradation of carotenoids during storage as the photostability of pigment is very much affected by its carotenoids’structure.
Colour properties analysis:
The fucoxanthin extract were evaluated with respect to lightness and chroma weekly during the four week storage period. Luminosity (L) is a measure of lightness or brightness of the colour. A positive ǻL value is lighter and a negative value is darker. Chroma, on the other hand, is a measure of colour intensity where the higher the value the higher is the intensity it represents (Diaz et al., 2006).  For instance, the higher luminosity value of sample indicated that the lower amount of pigment retained in the sample while the higher content of pigment was represented by higher chroma values.
In the present study, despite the method of treatments, all the samples stored in dark condition exhibited higher chroma values and smaller increase in lightness values when compared to the samples exposed to light (data not shown), indicating better stability of fucoxanthin with the absence of light source.
Table 1: Effect of ascorbic acid supplementation on retention of fucoxanthin
Ascorbic acid concentration (% w/v)Pigment retention (%)
----------------------------------------------------------------------------------------------
Light condition Dark condition
Control  2.949.1
0.1  5.655.9
0.510.166.4
1.07.871.0
Table 2: Effect of pH on retention of fucoxanthin
pH Pigment retention (%)
-----------------------------------------------------------------------------------------
Light condition Dark condition
3  1.729.3
5  1.937.3
7  2.048.7
9  2.162.1
Conclusion:
Fucoxanthin extracted from Sargassum binderi exhibited sensitivity towards different factors such as light, pH and addition of antioxidant. Overall, the fucoxanthin pigments were more resistant to degradation in dark condition as compared to pigment exposed to light which had accelerated rate of colour degradation. Addition of antioxidant further protects the fucoxanthin pigments from degradation. The fucoxanthin extract supplemented with 1.0% w/v of ascorbic acid displayed greatest pigment retention in both dark and light condition.  The most favourable pH condition in providing the greatest stability for the pigment was pH 9, especially in dark condition. Therefore, it could be concluded that the fucoxanthin pigments were sensitive to light exposure, the least stable in acidic pH condition and hi
gher concentration of ascorbic acid supplementation exerted stabilisation role on fucoxanthin.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge Department of Science, Faculty of Engineering and Science, Universiti Tunku Abdul Rahman (UTAR), Malaysia for the research facilities.
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