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Stability of polyhydroxylated 1,4

naphthoquinone pigment recovered from
spines of sea urchin Strongylocentrotus nudus
ARTICLE in INTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY JULY 2012
Impact Factor: 1.38 DOI: 10.1111/j.1365-2621.2012.02995.x

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International Journal of Food Science and Technology 2012, 47, 14791486

Original article
Stability of polyhydroxylated 1,4-naphthoquinone pigment
recovered from spines of sea urchin Strongylocentrotus nudus
Da-Yong Zhou,1 Bei-Wei Zhu,1* Xiao-Dong Wang,1 Lei Qin,1 Dong-Mei Li,1 Li Miao1 & Yoshiyuki Murata2
1 School of Food Science and Technology, Dalian Polytechnic University, Engineering Research Center of Seafood of Ministry of Education,
International Science & Technology Cooperation Base for Precious Seafood Deep Processing, Dalian 116034, China
2 Department of Biofunctional Chemistry, Division of Bioscience, Graduate School of Natural Science and Technology, Okayama University,
Okayama 700-8530, Japan
(Received 31 August 2011; Accepted in revised form 6 February 2012)

Summary

The stability of polyhydroxylated 1,4-naphthoquinone pigment recovered from sea urchin (Strongylocentrotus nudus) spines was observed by spectrophotometer. When pH values changed from 2.0 to 10.0, the
pigment showed colour variations. Meanwhile, pH values aected the stability of the pigment. After 24 h of
storage, the pigment remained 95.64%, 79.68% and 46.15% of the initial absorbance at pH 3.0, 7.0 and 10.0,
respectively. The pigment was prone to degrade on exposure to light. It remained 40.11% of the initial
absorbance under natural light after 14 days of storage. Whereas the corresponding value in the dark was
65.14%. Degradation rate of the pigment increased with increasing temperature. The pigment remained
90.49% of the initial absorbance at 20 C after 24 h of storage. However, the corresponding value decreased
to 66.61% at 80 C. Additives, including antioxidant, oxidising and reducing agents, and some metal ions all
aected the stability of the pigment.

Keywords

Pigment, polyhydroxylated 1,4-naphthoquinone, sea urchin (Strongylocentrotus nudus), spines, stability.

Introduction

Sea urchins belong to the marine invertebrate Echinoidea which live on the ocean oor. Some species of sea
urchins are valued for their gonads because they are
precious delicacies in many parts of the world. The shells
and spines of sea urchins are discarded as waste after
removal of gonads. As other food wastes, utilisation of
sea urchin shells and spines is necessary.
Food industry produces large volumes of wastes,
resulting from the production, preparation and consumption of food. Because of the high content of
organic components, proteins, oils and fats, and
polysaccharides, the environmental impact of these
wastes is generally high if disposed without suitable
treatment (Digman & Kim, 2008). To fully use of
resource and reduce environmental burden, numerous
researches have been carried out to develop methods
to convert these wastes into useful products (Arvanitoyannis, 2008). The utilisation methods and potential
uses of wastes from some types of food such as sh
(Arvanitoyannis & Kassaveti, 2008), meat (Arvanito*Correspondent: Fax: +86 0411 86323262;
e-mail: zhubeiwei@163.com

yannis & Ladas, 2008), grain (Arvanitoyannis &

Tserkezou, 2008a,b), olive oil (Arvanitoyannis et al.,
2007), dairy (Arvanitoyannis & Giakoundis, 2006) and
wine (Arvanitoyannis et al., 2006) have been well
summarised. These years, studies on utilisation of sea
urchin shells and spines have also been carried out.
To data, such type studies all focused on natural
pigments (Kuwahara et al., 2009, 2010; Zhou et al.,
2011).
In 1883, MacMunn rstly noticed the presence of
naphthoquinone pigments in a number of sea urchins
(Anderson et al., 1969). From then on, there are about
thirty naphthoquinone pigments have been identied
from sea urchin shells and spines (Anderson et al., 1969;
Utkina et al., 1976; Mischenko et al., 2005; Yakubovskaya et al., 2007). Those naphthoquinone pigments
almost all have the polyhydroxylated 1,4-naphthoquinone (PHNQ) structure. PHNQ pigments and their
synthetic analogues are known today as biologically
active compounds that possess high antimicrobial,
antialgal, antioxidant and cardioprotective activities
(Mischenko et al., 2005). Echinochrome A, a typical
PHNQ, has been used as the active ingredient in the
drug histochrome in Russia for preventing reperfusion
damages (Lebedev et al., 2005).

doi:10.1111/j.1365-2621.2012.02995.x
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Stability of pigments from sea urchin waste D.-Y. Zhou et al.

These years, natural pigments are being increasingly

emphasised owing to the fact that consumers perceive
them as safe additives contrary to their synthetic
counterparts (Sajilata & Singhal, 2006). It was estimated
that the natural pigments accounted for about 36.2% of
the global food pigments market in 2009 (Weston,
2010). Currently, the amount for permitted naturally
derived food pigments in EU and USA are 13 and 26,
respectively (Downham & Collins, 2000).
The stability of PHNQ pigments during processing
and storage is crucial to their use as food colorants.
Lebedev et al. (2001, 2003) have reported the eects of
pH and metal ions on the stability of echinochrome A
(6-ethyl-2,3,5,7,8-pentahydroxy-1,4-naphthoquinone).
Their studies indicated that auto-oxidation of echinochrome A accounted for its instability. Echinochrome A
solution was relatively stable in the neutral and acid pH
ranges. Some metal ions such as Ca2+decreased the
stability of echinochrome A by increasing the autooxidation rate. However, information of other main
factors inuencing colour stability such as heat, light
and food antioxidants is still unavailable.
In our previous study, the PHNQ pigments were
prepared from purple sea urchin (Strongylocentrotus
nudus) spines using macroporous resin in static adsorption mode (Zhou et al., 2011). The pigments were
characterised rapidly by using an ultra-performance
liquid chromatography (UPLC) coupled to hybrid
quadrupole orthogonal acceleration time-of-ight mass
spectrometer (Q-TOFMS). Six known compounds,
including Spinochrome E, 2,7-dihydroxynaphthazarin,
Spinochrome B, Spinochrome C, Spinochrome A,
Echinochrome A, and two new compounds with molecular formula of aminopentahydroxynaphthoquinone
and acetylaminotrihydroxynaphthoquinone were identied tentatively. The objective of this study was to
investigate the eect of pH, light, temperature, vitamin
C, oxidising and reducing agents, and metal ions on the
stability of the PHNQ pigments.
Material and methods

initial volume. The concentrate was diluted to three

times of its initial volume with deionised water and
centrifuged at 1200 g for 10 min. One litre of the
supernatant was loaded onto a glass column
(10 40 cm) wet-packed with 500 g of NKA-9 macroporous resin (The Chemical Plant of Nankai University,
Tianjin, China) and let stand at room temperature for
3 h (the bottom outlet of the column was closed). After
emptying of liquid, the column was washed thoroughly
with 3 L of deionised water and eluted with 2 L of 95%
ethanol aqueous solution. The eluate was collected,
lyophilised using a freeze dryer (2KBTES-55; VirTis
Co., Gardiner, NY, USA) and stored at )20 C in the
dark until use.
One gram of pigment was accurately weighed, dissolved in 250 mL of deionised water and centrifuged at
9300 g for 10 min. The supernatant, which was always
prepared just before use, was used as a working
solution.
Ultravioletvisible (UVVis) spectroscopy of pigment

Twenty-ve micrograms of pigment was dissolved in

50 mL of deionised water or methanol and centrifuged
at 9300 g for 10 min. The UV Vis spectrum of the
supernatant was recorded at 200700 nm using an
UV Visible spectrophotometer (LAMBDA 35; Perkin
Elmer Co., Waltham, MA, USA).
Influence of pH on the colour and stability of pigment

Pigment working solution was diluted to ten times of its

initial volume with deionised water and was adjusted to
dierent pH values (pH 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0
and 10.0) with 3 m HCl and NH3OH. The UV Vis spectra
of the pigment solutions with various pH values were
recorded at 200700 nm using an UV Visible spectrophotometer (LAMBDA 35, Perkin Elmer Co). The
pigment solutions with various pH values were placed
in 10 mL of sealed glass tubes and left in the dark at room
temperature (17 3 C). The absorbance of the pigment
solution at 475 nm was read at 0, 1, 2, 3, 5, 7 and 24 h.

Preparation of PHNQ pigment

Sea urchin (Strongylocentrotus nudus) was collected

from Yellow sea, China, in July 2009. The spines were
collected, washed with a stream of cold water, dried at
40 C in the dark using a drying oven and then
grounded into powder. Seven hundred grams of the
powder was added gradually to 5 L of ethanolHCl
aqueous solution (95% ethanol (v): 37% HCl (v) = 7:3)
with continuous stirring. After dissolution, the solution
was stirred continuously for another more 10 min.
Following centrifugation at 1200 g for 10 min, the
supernatant was collected and concentrated using a
rotary vacuum evaporator at 45 C to a quarter of its

International Journal of Food Science and Technology 2012

Influence of light on the stability of pigment

Pigment working solution was diluted to four times of

its initial volume with deionised water and stored at
room temperature (17 3 C) in the dark or under
natural light. The absorbance of the pigment solution at
475 nm was read at 0, 1, 2, 3, 5, 7 and 14 days.
Influence of temperature on the stability of pigment

Pigment working solution was diluted to four times of

its initial volume with deionised water, placed in 10 mL
of sealed glass tube and left in the dark at 20, 40 and

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Stability of pigments from sea urchin waste D.-Y. Zhou et al.

80 C. The absorbance of the pigment solution at

475 nm was read at 0, 1, 2, 3, 5, 7 and 24 h.

(a) 4.5
4

Influence of additive agents on the colour and stability of

pigment

Absorbance

One volume of pigment working solution mixed with

nine volumes of various concentrations of vitamin C (0,
0.01, 0.05, 0.1, 0.5 and 1.0 g L)1), hydrogen peroxide (0,
0.2, 0.4, 0.6, 0.8 and 1.0%), sodium sulphite (0, 0.1, 1.0,
1.5, 2.0 and 5.0 g L)1) and metal ions such as Mn2+,
Fe2+, Fe3+, Cu2+, Ca2+, Zn2+, Mg2+and Al3+ (0, 2,
4, 6, 8 and 10 mm). The pigment solutions containing
various concentrations of additive agents were placed in
10 mL of sealed glass tubes and left in the dark at room
temperature (17 3 C). The absorbance of the pigment solution at 475 nm was read at 0, 1, 2, 3, 5, 7 and
14 days (for vitamin C) or 0, 1, 2, 3, 5, 7 and 24 h (for
other additive agents except for vitamin C).

3.5

2.5

Methanol

pH 5

pH 2

pH 6

pH 3

pH 7

pH 4

2
1.5
1
0.5
0
220

280

340

400

460

520

580

640

700

580

640

700

Wavelength (nm)

One volume of pigment working solution was mixed with

nine volumes of 0.1 g L)1 vitamin C or deionised water.
The pigment solutions were placed in 10 mL of sealed
glass tubes and left in the dark at room temperature
(17 3 C). After 0, 2 and 4 h of storage time, the
pigment solutions were sampled, ltered through a 0.45lm membrane lter and injected to a HPLC system for
analysis. The HPLC system used for this study was a
Waters 2695 Separations Module coupled to a Waters
2998 Photodiode Array Detector (Waters, Milford, MA,
USA). The analytical column was a SunFire C18 column
(150 mm4.6 mm, 5 lm; Waters). The two solvents were
phase A: methanol; phase B: water acetic acid (v v,
100 0.3). The solvents were ltered through a 0.45-lm
membrane lter unit prior to using. A linear gradient was
programmed: 010 min: 5% B (v v); 1040 min: 590%
B; 4041 min: 9095% B; 4150 min: 95% B. The ow
rate was 1 mL min)1. The injection volume was 10 lL.
Statistic method

All the tests were conducted with three replicates. Data

were presented as means standard deviations. Dierences between means were evaluated by least-signicant
dierence test of analysis of variance (anova). The
statistical analysis was performed using SPSS 15.0
software (SPSS Inc. Chicago, IL, USA). Comparisons
that yielded P values <0.05 were considered signicant.
Results and discussion

The pigment extract prepared from Strongylocentrotus

nudus spines is a mixture including eight PHNQ

(b) 4.5
4
3.5
3

Absorbance

Analysis of influence of vitamin C on the stability of

pigment using HPLC

pH 8
2.5

pH 9
pH 10

2
1.5
1
0.5
0
220

280

340

400

460

520

Wavelength (nm)
Figure 1 UV Vis spectra of the pigment in methanol solution and
aqueous solutions with various pH values ((a) methanol solution and
aqueous solutions of pH 27; (b) aqueous solutions of pH 810).

compounds and showed orange-red in colour in aqueous solution (Zhou et al., 2011). Therefore, the UV Vis
spectrum of the pigment is an overlay of the optical
absorption of all the compounds. As shown in Fig. 1a,
the spectrum of the pigment in aqueous solution (the pH
of the pigment aqueous solution was about 4.0) showed
typical features of PHNQ: the benzenoid electron
transfer (ET) bands are centred at 247 and 475 nm;
the quinoid ET bands are centred at 265 and 302 nm

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International Journal of Food Science and Technology 2012

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Stability of pigments from sea urchin waste D.-Y. Zhou et al.

(Singh et al., 1968). The pigment in methanol solution

showed a slightly dierent UV Vis spectrum in comparison with that in water: the band around 247 nm
disappeared and the band around 302 nm shifted
bathochromically to 320 nm. The maximum absorbent
wavelength at about 475 nm could be chosen for PHNQ
stability studies because of its high specicity for this
type pigments.
Polyhydroxylated 1,4-naphthoquinone have several
acid dissociation constants. For example, the dissociation constants such as pKa1, pKa2 and pKa3 of
echinochrome A (6-ethyl-2,3,5,7,8-pentahydroxy-1,4naphthoquinone) are 5.20, 6.78 and >10 (Lebedev
et al., 2003). Therefore, PHNQ will be present in their
un-ionised or anion forms in aqueous solution with
dierent pH values. It was reported that the state of
dissociation of PHNQ can aect their colour (Singh
et al., 1968) and stability (Lebedev et al., 2001, 2003).
Figure 1 showed the UVVis spectra of the pigment in
aqueous solutions with pH values ranged from 2.0 to
10.0. When pH values ranged from 2.0 to 6.0, the
UVVis spectra of the pigment in aqueous solutions
showed the diagnostic features of such type compounds
as described earlier. At this range of pH values, PHNQ
are present mainly in their un-ionised forms. When pH
values ranged from 7 to 8, the bands at 247 and 265 nm
shifted bathochromically to 287 and 365 nm, and the
band at 475 nm diminished. Meanwhile, the colour of
the pigment solutions changed from orange-red to
earth-yellow. At this range of pH values, the monoand divalent anion forms of PHNQ are formed because
of the dissociation of b-hydroxyl groups (Lebedev et al.,
2003). It was reported that the anionic b-hydroxyls of
PHNQ can result in small bathochromic shift at UV
region (Singh et al., 1968). When pH values ranged from
9 to 10, the bands at 287 and 365 nm diminished, and
the band at 475 nm shifted bathochromically to 560
580 nm. In addition, the pigment aqueous solutions

showed deep-earth-yellow. Polyvalent anion forms of

PHNQ may be formed at dissociation of hydroxyl
groups on the benzenoid at pH above 9 (Lebedev et al.,
2003). Singh et al. (1968) reported that the anionic form
of the peri-hydroxyl group produces a large bathochromic shift of the visible band. The naphthoquinone
pigments isolated from the roots of Lithospermum
erythrorhizon showed the similar UVVis spectral variations at dierent pH values (Cho et al., 1999).
Additionally, the pH variation aects appreciably the
stability of the pigment in aqueous solution. The
pigment in aqueous solution showed the highest stability
at strongly acidic conditions (pH 2.03.0), the modest
stability at weak acidic to neutral conditions (pH 4.0
8.0) and the lowest stability at basic conditions (pH 9.0
10.0) (Table 1). At basic conditions, the di- and polyvalent anion forms of PHNQ are formed owing to the
dissociation of hydroxyl group (Lebedev et al., 2001). In
polyvalent anion forms, PHNQ are sensitive to oxygen,
and auto-oxidation can readily take place (Lebedev
et al., 2001, 2003). Through auto-oxidation, PHNQ
changed into naphthosemiquinone and naphthotetraketone and lose the initial absorbance at 475 nm (Lebedev et al., 2003). Auto-oxidation is one of the main
reasons for the instability of PHNQ in processing and
storage.
As shown in Table 2, the absorbance (475 nm) of the
pigment in aqueous solution increased at initial stage
and decreased gradually thereafter in the presence of
vitamin C, indicating that vitamin C can protect and
enhance the colour of PHNQ pigment. As an antioxidant, vitamin C can prevent the auto-oxidation of
PHNQ pigment, which may account for the colour
protecting eect. To reveal the reason why vitamin C
can enhance the colour (absorbance at 475 nm) of
PHNQ pigment, we analysed the pigment aqueous
solution with and without vitamin C using HPLC.
Contrasted to 0 h, the peak area of Spinochrome B

Table 1 Effect of pH values on stability of the polyhydroxylated 1,4-naphthoquinone (PHNQ) pigment

Storage time (h)
pH

2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0

97.57
98.61
98.10
96.53
90.34
82.70
94.13
82.20
65.28

1.35abA1-3
0.96aA
1.94abA
0.97bA
2.70dA
1.00eA
2.62cA
0.69eA
1.52fA

96.74
97.05
97.01
91.41
81.37
80.41
87.58
68.97
56.48

1.22aA
2.50aAB
2.41aA
1.58bB
1.22dB
3.19dAB
3.24cB
3.53eB
2.65fB

96.40
95.63
93.23
85.23
75.64
81.79
85.58
61.07
51.27

1.62aA
1.82abB
3.15bB
2.26cC
2.05eC
1.60dAB
2.18cBC
2.19fC
2.35gC

96.49
95.26
87.30
83.85
76.40
81.75
82.82
52.95
48.06

1.84aA
1.21aB
2.66bC
4.81cCD
2.98dC
1.39cAB
1.93cCD
3.36eD
0.98fD

96.33
95.59
83.27
80.64
75.52
81.38
80.75
49.15
47.86

24

0.49aA
2.43aB
0.82bD
3.91bD
3.94cC
2.53bAB
2.77bDE
1.18dE
1.29dD

96.82
95.64
76.06
76.24
73.16
79.68
79.65
48.92
46.15

2.53aA
2.77aB
2.07cE
3.85cE
3.23cC
2.65bB
2.81bE
2.20dE
1.01dD

Mean values within a column with different lower case letters are significantly different at P < 0.05.
Mean values within a row with different upper case letters are significantly different at P < 0.05.
3
The values represent the percentage of the initial absorbance.
2

International Journal of Food Science and Technology 2012

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International Journal of Food Science and Technology  2012 Institute of Food Science and Technology

Stability of pigments from sea urchin waste D.-Y. Zhou et al.

Table 2 Effect of vitamin C concentrations on stability of sea urchin pigment

Vitamin C concentrations (g L)1)
Time (days)

1
2
3
5
7
14

87.78
81.25
77.95
72.00
68.58
51.32

0.009

1.85cA1-3
2.11cB
3.30cB
3.12dC
3.40cC
3.69cD

113.17
113.97
120.06
118.70
120.02
110.92

0.045

4.28abB
1.47bB
0.40bA
3.36bcAB
7.77bA
5.57bB

111.62
117.45
119.53
122.67
127.46
121.37

0.09

2.83bD
3.02bC
0.30bBC
4.22bcB
3.95abA
1.88aB

0.45

118.82
125.08
131.00
129.69
132.98
128.31

3.43aD
1.77aC
3.06aAB
3.32aABC
3.43aA
8.14aBC

115.89
117.83
126.85
123.91
126.59
122.35

0.9

4.92abD
3.32bCD
1.28aA
2.25abAB
0.90abA
0.48aBC

113.79
116.50
119.15
117.59
117.97
110.96

1.32abA
3.13bA
3.68bA
4.13cA
3.58bA
0.44bA

Mean values within a row with different lower case letters are significantly different at P < 0.05.
Mean values within a column with different upper case letters are significantly different at P < 0.05.
3
The values represent the percentage of the initial absorbance.
2

(Zhou et al., 2011), which was the main PHNQ

compound in the pigment solution, was increased by
15.40% and 19.05% with storage time of 2 and 4 h with
the addition of vitamin C. Whereas the peak area of
Spinochrome B was decreased by 0.90% and 0.40%
with the comparative storage time without the addition
of vitamin C. It was speculate that a part of PHNQ
compounds in the pigment extract has been oxidised
during the process of isolation and concentration. As an
electron donor, vitamin C is a reducing agent (Padayatty
et al., 2003). Therefore, vitamin C can reduce the
oxidised PHNQ compounds to their initial structures,
which may contribute to the colour enhancing eect.
Meanwhile, results indicated that the colour protecting
and enhancing eect of vitamin C varied with various
concentrations (Table 2). It was interested that the
colour protecting and enhancing eect did not increased
continuously with increasing vitamin C concentration.
Whereas the eect decreased when vitamin C concentration was more than 0.09 g L)1. Actually, vitamin C
can serve both as a pro-oxidant and as an antioxidant
(Yen et al., 2002). We speculated that a part of vitamin
C may act as pro-oxidant at high concentrations and has
an adverse eect on colour protecting and enhancing
eect.
As shown in Fig. 2a, the degradation of the pigment
was more pronounced under natural light in comparison
with in the dark. After 14 days of storage, the pigment
solution remained 40.11% and 65.14% of the initial
absorbance under natural light and in the dark. Cho
et al. (1999) observed that the 1,4-naphthoquinone
pigments isolated from the roots of Lithospermum
erythrorhizon showed photodegradation. Their ndings
suggested that the substituted groups do not aect the
photolysis of the 1,4-naphthoquinone. As shown in
Fig. 2b, the stability of the pigment in aqueous solution
decreased with increasing temperature. After 24 h of
storage, the pigment solution remained 90.49%, 78.42%
and 66.61% of the initial absorbance at 20, 40 and
80 C, respectively. The 1,4-naphthoquinone pigments
isolated from the roots of Lithospermum erythrorhizon

also showed thermal degradation (Cho et al., 1999). In

contrast to photodegradation, the heat stabilities of the
1,4-naphthoquinone pigments were dierent with substituted groups.
PHNQ possess both the properties of electron donor
and of electron acceptor. Thus, these compounds may
be either reduced cathodically or oxidised anodically
(Petrova et al., 1995). The pigment aqueous solution
became earth-yellow by adding Na2SO3, a common
reducing agent. Meanwhile, Na2SO3 decreased the
stability of the pigment in aqueous solution in a
dose-dependent manner (Fig. 3a). Moore et al. (1966)
reported that 1,4-naphthoquinone are presumably
reduced to 1,4-naphthalene by bonding of two hydrogen
atoms to the carbonyl oxygen in the presence of
reducing agent. In addition, the aqueous solution of
Na2SO3 is weakly basic. In basic condition, PHNQ are
sensitive to oxygen (Lebedev et al., 2001, 2003). By
contrast, the addition of H2O2, a common oxidising
agent, did not change the colour of the pigment aqueous
solution. However, H2O2 decreased the stability of the
pigment in aqueous solution in a dose-dependent
manner (Fig. 3b). Kuwahara et al. (2009) reported that
PHNQ can scavenge hydrogen peroxide. As electron
donor, PHNQ may be oxidised to naphthosemiquinone
and naphthopolyketone.
The inuence of metal ions including Mn2+, Mg2+,
Ca2+, Zn2+, Al3+, Fe3+, Fe2+and Cu2+on colour and
stability of PHNQ pigment varied with the types of
metal ions. Metal ions, including Mn2+and Mg2+, did
not aect the colour and the stability of the pigment in
aqueous solution at concentrations below 9 mm
(Fig. 4a, b). Lebedev et al. (2003) also reported that
the two metal ions had no eect on the auto-oxidation
rate of echinochrome A.
Metal ions, including Ca2+and Zn2+, decreased the
stability but did not aect the colour of the pigment in
aqueous solution at concentrations below 9 mm (Fig. 4c,
d). Lebedev et al. (2003, 2001) observed that addition of
Ca2+to echinochrome A aqueous solution led to an
increase in auto-oxidation rate. Their ndings suggested

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Stability of pigments from sea urchin waste D.-Y. Zhou et al.

(a) 100

(a) 110
100

90

80

% Initial absorbance

% Initial absorbance

90

70

60

50

80
70
60
50

Light
40

30

Dark

40
30

10

12

14

1.35

0.09

1.8

0.9

4.5

(b) 110

95

100

90

90

% Initial absorbance

(b) 100

85
80
75
70

20 C

65

80 C

24

24

60

16

20

24

Time (h)

0.54
0.72
0.90

0
0.18
0.36

40

12

70

30
0

80

50

40 C

60

Storage time (h)

Time (days)

% Initial absorbance

1484

Storage time (h)

)1

Figure 2 Effect of light (a) and temperature (b) on stability of the

polyhydroxylated 1,4-naphthoquinone (PHNQ) pigment in aqueous

solution.

that complexing of Ca2+leads to a decrease in the

dissociation constant of PHNQ. Thus, PHNQ are prone
to present in polyvalent anion forms in aqueous solution
in the presence of Ca2+. In polyvalent anion forms,
PHNQ are sensitive to oxygen and are readily autooxidised (Lebedev et al., 2001, 2003). Their ndings also
suggested that addition of Ca2+to echinochrome A
aqueous solution led to the formation of stable calciumsemiquinone adducts (Lebedev et al., 2001). Semiquinon-

International Journal of Food Science and Technology 2012

Figure 3 Effect of various concentrations (g L ) of (a) Na2SO3 and

(b) H2O2 on stability of the polyhydroxylated 1,4-naphthoquinone

(PHNQ) pigment in aqueous solution.

es are important intermediates of oxidation reaction of

PHNQ. Formation of the calcium-semiquinone adducts
can shift the oxidationreduction equilibrium to the left
(oxidation).
The pigment aqueous solution became black-red by
adding Al3+and became tan by adding Fe3+. However,
the addition of the two metal ions did not cause
precipitation. The change in colour of the pigment
aqueous solution may be due to the formation of

 2012 The Authors

International Journal of Food Science and Technology  2012 Institute of Food Science and Technology

(a) 110

(c) 110

(e) 110

% Initial absorbance

% Initial absorbance

% Initial absorbance

Stability of pigments from sea urchin waste D.-Y. Zhou et al.

100

90

80

70

0
1.8

5.4
7.2

3.6

9.0

100

90

80

70

24

(d) 110

% Initial absorbance

% Initial absorbance

100

90

70

5.4
7.2

3.6

9.0

Storage time (h)

3.6

9.0
2

24

Storage time (h)

(b) 110

0
1.8

5.4
7.2

Storage time (h)

80

0
1.8

100

24

90

80

70

0
1.8

5.4
7.2

3.6

9.0

24

Storage time (h)

100

90

80

70

0
1.8

5.4
7.2

3.6

9.0
2

24

Storage time (h)

Figure 4 Effect of various concentrations (mm) of metal ions on stability of the polyhydroxylated 1,4-naphthoquinone (PHNQ) pigment in aqueous
solution ((a) Mn2+; (b) Mg2+; (c) Ca2+; (d) Zn2+; (e) Al3+).

water-soluble metal ionPHNQ complex (Lebedev et al.,

2005). The stability of the complexes is decreased
(Fig. 4e) as the metal ionPHNQ complexes are more
sensitive to oxygen (Lebedev et al., 2001, 2003). The
addition of Fe2+to the pigment aqueous solution caused
a black precipitate immediately. The pigment aqueous
solution became yellowish-green immediately by adding Cu2+. After a while, a reddish-brown precipitate
occurred. After centrifugation, the both supernatants
were colourless. Lebedev et al. (2005) also observed the
precipitation by adding Fe2+to echinochrome A aqueous
solution. The formation of water-insoluble metal ion
PHNQ complexes may account for the precipitation.
Conclusion

In this study, the stability of PHNQ pigment recovered

from Strongylocentrotus nudus spines during storage was
investigated. The pigment was prone to degrade on
exposure to light and hot. Consequently, it should be
stored in a dark and low-temperature condition. Some
factors, including alkaline condition, oxidising agents
and some metal ions such as Ca2+, Zn2+, Al3+and
Fe3+, may increase the auto-oxidation rate of the
pigment. Meanwhile, some metal ions such as Fe2+and

Cu2+can cause the precipitation of the pigment. Therefore, the pigment should be stored and applied under the
conditions without those adverse factors. In contrast,
antioxidant such as vitamin C can protect and enhance
the colour of the pigment, so the pigment should be
stored and applied along with some antioxidants.
Acknowledgments

This work was nancially supported by The National

High Technology Research and Development Program
of China (863 Program) (No. 2011AA100803) and The
Research Start-up Project for Doctor Funded by Liaoning Science and Technology Department (No.
20091002).
References
Anderson, H.A., Mathieson, J.W. & Thomson, R.H. (1969). Distribution of spinochrome pigments in echinoids. Comparative Biochemistry and Physiology, 28, 333345.
Arvanitoyannis, I.S. (2008). Waste Management for the Food Industries. Amsterdam, Netherlands: Elsevier.
Arvanitoyannis, I.S. & Giakoundis, A. (2006). Current strategies for
dairy waste management: a review. Critical Reviews in Food Science
and Nutrition, 46, 379390.

 2012 The Authors

International Journal of Food Science and Technology  2012 Institute of Food Science and Technology

International Journal of Food Science and Technology 2012

1485

1486

Stability of pigments from sea urchin waste D.-Y. Zhou et al.

Arvanitoyannis, I.S. & Kassaveti, A. (2008). Fish industry waste:

treatments, environmental impacts, current and potential uses.
International Journal of Food Science and Technology, 43, 726745.
Arvanitoyannis, I.S. & Ladas, D. (2008). Meat waste treatment
methods and potential uses. International Journal of Food Science
and Technology, 43, 543559.
Arvanitoyannis, I.S. & Tserkezou, P. (2008a). Wheat, barley and oat
waste: a comparative and critical presentation of methods and
potential uses of treated waste. International Journal of Food Science
and Technology, 43, 694725.
Arvanitoyannis, I.S. & Tserkezou, P. (2008b). Corn and rice waste: a
comparative and critical presentation of methods and current and
potential uses of treated waste. International Journal of Food Science
and Technology, 43, 8988.
Arvanitoyannis, I.S., Ladas, D. & Mavromatis, A. (2006). Wine waste
treatment methodology. International Journal of Food Science and
Technology, 41, 11171151.
Arvanitoyannis, I.S., Kassaveti, A. & Stefanatos, S. (2007). Current
and potential uses of thermally treated olive oil waste. International
Journal of Food Science and Technology, 42, 852867.
Cho, M.H., Paik, Y.S. & Hahn, T.R. (1999). Physical stability of
shikonin derivatives from the roots of Lithospermum erythrorhizon
cultivated in Korea. Journal of Agricultural and Food Chemistry, 47,
41174120.
Digman, B. & Kim, D.S. (2008). Review: alternative energy from food
processing wastes. Environmental Progress, 27, 524537.
Downham, A. & Collins, P. (2000). Colouring our foods in the last and
next millennium. International Journal of Food Science and Technology, 35, 522.
Kuwahara, R., Hatate, H., Yuki, T., Murata, H., Tanaka, R. & Hama,
Y. (2009). Antioxidant property of polyhydroxylated naphthoquinone pigments from shells of purple sea urchin Anthocidaris
crassispina. Lwt-Food Science and Technology, 42, 12961300.
Kuwahara, R., Hatate, H., Chikami, A., Murata, H. & Kijidani, Y.
(2010). Quantitative separation of antioxidant pigments in purple
sea urchin shells using a reversed-phase high performance liquid
chromatography. Lwt-Food Science and Technology, 43, 11851190.
Lebedev, A.V., Levitskaya, E.L., Tikhonova, E.V. & Ivanova, M.V.
(2001). Antioxidant properties, autooxidation, and mutagenic
activity of echinochrome A compared with its etheried derivative.
Biochemistry (Moscow), 66, 885893.
Lebedev, A.V., Ivanova, M.V. & Ruuge, E.K. (2003). How do calcium
ions induce free radical oxidation of hydroxy-1,4-naphthoquinone?

International Journal of Food Science and Technology 2012

Ca2+ stabilizes the naphthosemiquinone anion-radical of echinochrome A Archives of Biochemistry and Biophysics, 413, 191198.
Lebedev, A.V., Ivanova, M.V. & Levitsky, D.O. (2005). Echinochrome, a naturally occurring iron chelator and free radical
scavenger in articial and natural membrane systems. Life Sciences,
76, 863875.
Mischenko, N.P., Fedoreyev, S.A., Pokhilo, N.D., Anufriev, V.P.,
Denisenko, V.A. & Glazunov, V.P. (2005). Echinamines A and B,
rst aminated hydroxynaphthazarins from the sea urchin Scaphechinus mirabilis. Journal of Natural Products, 68, 13901393.
Moore, R.E., Singh, H., Chang, C.W.J. & Scheuer, P.J. (1966).
Sodium borohydride reduction of spinochrome A. removal of
phenolic hydroxyls in the naphthazarin system. The Journal of
Organic Chemistry, 31, 36383645.
Padayatty, S.J., Katz, A., Wang, Y. et al. (2003). Vitamin C as an
antioxidant: evaluation of its role in disease prevention. Journal of
the American College of Nutrition, 22, 1835.
Petrova, S.A., Ksenzhek, O.S. & Kolodyazhny, M.V. (1995). Redox
properties of echinochrome A. Journal of Electroanalytical Chemistry, 384, 131137.
Sajilata, M.G. & Singhal, R.S. (2006). Isolation and stabilisation of
natural pigments for food applications. Stewart Postharvest Review,
2, 129.
Singh, I., Ogata, R.T., Moore, R.E., Chang, C.W.J. & Scheuer, P.J.
(1968). Electronic spectra of substituted naphthoquinones. Tetrahedron, 24, 60536073.
Utkina, N.K., Shchedrin, A.P. & Maksimov, O.B. (1976). A new
binaphthoquinone from Strongylocentrotus intermedius. Chemistry
of Natural Compounds, 12, 387389.
Weston, S. (2010).Food colouring a global overview. [Internet
document] URL http://www.foodbev.com/article/food-colouring-aglobal-overview. Accessed 08 11 2010.
Yakubovskaya, A.Y., Pokhilo, N.D., Mishchenko, N.P. & Anufriev,
V.F. (2007). Spinazarin and ethylspinazarin, pigments of the sea
urchin Scaphechinus mirabilis. Russian Chemical Bulletin, 56, 819
822.
Yen, G.C., Duh, P.D. & Tsai, H.L. (2002). Antioxidant and prooxidant properties of ascorbic acid and gallic acid. Food Chemistry,
79, 307313.
Zhou, D.Y., Qin, L., Zhu, B.W. et al. (2011). Extraction and
antioxidant property of polyhydroxylated naphthoquinone pigments from spines of purple sea urchin Strongylocentrotus nudus.
Food Chemistry, 129, 15911597.

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International Journal of Food Science and Technology  2012 Institute of Food Science and Technology