Food Chemistry 129 (2011) 1591–1597

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Extraction and antioxidant property of polyhydroxylated naphthoquinone

pigments from spines of purple sea urchin Strongylocentrotus nudus
Da-Yong Zhou a, Lei Qin a, Bei-Wei Zhu a,⇑, Xiao-Dong Wang a, Hui Tan a, Jing-Feng Yang a, Dong-Mei Li a,
Xiu-Ping Dong a, Hai-Tao Wu a, Li-Ming Sun a, Xiu-Ling Li b, Yoshiyuki Murata c
a
School of Food Science and Technology, Dalian Polytechnic University, Engineering Research Center of Seafood, Ministry of Education, Liaoning Key Laboratory of Seafood Science
and Technology, Dalian 116034, PR China
b
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, PR China
c
Department of Biological Resources Chemistry, Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan

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

Article history: Purple sea urchin (Strongylocentrotus nudus) spines were dissolved in HCl–ethanol-aqueous solution. The
Received 22 May 2010 polyhydroxylated 1,4-naphthoquinone (PHNQ) pigments were condensed and purified by using macro-
Received in revised form 21 May 2011 porous resin in static adsorption mode. PHNQ in the extract were characterised rapidly by using an
Accepted 13 June 2011
ultra-performance liquid chromatography (UPLC) coupled to hybrid quadrupole orthogonal acceleration
Available online 16 June 2011
time-of-flight mass spectrometer (Q-TOFMS). Six known compounds including spinochrome E, 2,7-
dihydroxynaphthazarin, spinochrome B, spinochrome C, spinochrome A and echinochrome A, and two
Keywords:
new compounds with molecular formula of aminopentahydroxynaphthoquinone and acety-
Sea urchin
Natural pigment
laminotrihydroxynaphthoquinone were identified tentatively. The pigment extract was evaluated for
Naphthoquinone antioxidant activity by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging capacity assay, Fe2+ che-
Extraction lating assay, reducing power assay, lipid peroxidation inhibition assay and tertiary-butyl hydroperoxide
Antioxidant (t-BOOH)-induced macrophages protection assay. In all testing methods, the extract showed excellent
activity, indicating the PHNQ from S. nudus spines are potential sources of natural antioxidants.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction polyhydroxylated 1,4-naphoquinone (PHNQ) structure with ethyl,

Sea urchin belongs to the marine invertebrate Echinoidea live on
the ocean floor. Hitherto, more than 800 species of sea urchin have
been found. The edible portion of sea urchin, gonad, is half-moon
shaped, yellow-orange in colour, and accounts for approximately
10% of the total weight (De la Cruz-Garcia, Lopez-Hernandez,
Gonzalez-Castro, De Quiros, & Simal-Lozano, 2000). With distinc-
tive aroma and good taste, sea urchin gonad is popular in many
countries.
The shells and spines are the major processing by-products of
sea urchin and their value-added utilisation is necessary (Kuwaha-
ra et al., 2009). In 1938, Lederer and Glaser identified the first qui-
nonoid pigment (spinochrome A) from sea urchin (Paracentrotus
lividus) spines (Goodwin & Srisukh, 1950). From then on, there
are about thirty quinonoid pigments have been isolated from sea
urchin of different species (Amarowicz, Synowiecki, & Shahidi,
1994; Anderson, Mathieson, & Thomson, 1969; Kol’tsova, Deni-
senko, & Maksimov, 1978; Mischenko et al., 2005; Utkina, Shche-
drin, & Maksimov, 1976; Yakubovskaya, Pokhilo, Mishchenko, &
Anufriev, 2007). Those quinonoid pigments almost all have the
⇑ Corresponding author. Fax: +86 0411 86323262.
E-mail address: zhubeiwei@163.com (B.-W. Zhu).
acetyl, methoxy and amino as substitutes (Amarowicz et al.,
1994; Anderson et al., 1969; Kol’tsova et al., 1978; Mischenko
et al., 2005; Utkina et al., 1976; Yakubovskaya et al., 2007). In re-
cent years, the PHNQ pigments were found to possess excellent
antimicrobial, antialgal, cardioprotective and antioxidant activities
(Mischenko et al., 2005). In Russia, echinochrome A, a typical
PHNQ, has been used as the active substance in the new drug ‘‘his-
tochrome’’ for preventing reperfusion damages developing during
the treatment of myocardial infarction by a thrombolytic (Lebedev,
Ivanova, & Levitsky, 2005). This suggests that sea urchin shells and
spines, most of which have been dumped as waste after removal of
gonads, may serve as a new biologically active resource.
PHNQ pigments are packaged in calcareous skeleton in sea
urchin shells and spines. Usually, the shells and spines are dis-
solved in HCl-aqueous solution, and then the PHNQ pigments are
extracted by organic solvents such as diethyl ether and chloroform
(Amarowicz et al., 1994; Kol’tsova et al., 1978; Mischenko et al.,
2005; Utkina et al., 1976). Those organic solvents are toxic and vol-
atile, indicating the operation is harmful to the operators and the
environment. Therefore, a green and safe extraction method is nec-
essary. It has been reported that the bioactivity of PHNQ depend on
their substituents (Lebedev et al., 2005). For example, the hydroxyl
substituents in the 2-, 3- and 7-positions are key structural factors

0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.06.014
1592 D.-Y. Zhou et al. / Food Chemistry 129 (2011) 1591–1597
for the antioxidant activity of PHNQ (Lebedev et al., 2005). Mean-
while, study indicated the predominant PHNQ are different in dif-
ferent species of sea urchin (Anderson et al., 1969). Therefore, the
rapid characterisation of PHNQ in a sea urchin species which has
not been studied is important as it may foretell whether those
PHNQ deserve further research. However, there is still no method
on rapid characterisation of PHNQ in crude samples.
Purple sea urchin, Strongylocentrotus nudus, is one of the most
popular edible species in China and Japan, and is very plentiful
on the coasts of Dalian (Huang-Bo Sea). In the present study, the
PHNQ pigments from S. nudus spines were extracted by using mac-
roporous adsorption resin and identified tentatively by using UPLC/
Q-TOFMS. Meanwhile, the antioxidant ability of the pigment ex-
tract was tested by using DPPH scavenging capacity assay, Fe2+
chelating assay, reducing power assay, lipid peroxidation inhibi-
tion assay and t-BOOH-induced macrophages protection assay.
2. Materials and methods
2.1. Chemicals
Macroporous resins D4020, D101, NKA-9, NKA-II and AB-8 were
purchased from The Chemical Plant of Nankai University (Tianjin,
China). DPPH and t-BOOH were purchased from Sigma Chemical
Co. (St Louis, MO, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) was purchased from Ameresco Co. (So-
lon, OH, USA). Dimethyl sulphoxide (DMSO) was purchased from
Beijing Solarbio Science and Technology Co. (Beijing, China). All
other chemicals and solvents used in this study were of analytical
or HPLC grade.
2.2. Preparation of crude pigments from sea urchin spines
Sea urchin (S. nudus) was collected from Yellow sea, China, from
May to August, in 2008. The spines were collected, washed with a
stream of cold water, freeze-dried in dark, and then ground into
powder. Fifty grams of powder were dissolved by gradually adding
1000 ml of ethanol–HCl-aqueous solution (95% ethanol (v/v):37%
HCl (v/v) = 7:3). Following centrifugation at 2000g for 10 min, the
supernatant was dried by rotary vacuum evaporator at 45 °C to
get the crude pigment extract.
2.3. Condensation and purification of pigments by macroporous resin
Five types of macroporous resins including D4020, D101, NKA-
9, NKA-II and AB-8 were used in this study. The macroporous res-
ins were pretreated according to the manufacturer’s instructions.
The adsorption and desorption procedure was performed as de-
scribed by Wang, Li, Zeng, and Liu (2008), with some modifications.
The crude pigment extract was dissolved in 0.4 M HCl to reach
an absorbance of 0.8–1.0 at 475 nm. Twenty millilitres of pigment
solution was introduced into a 100 ml conical flask containing 2.0 g
of macroporous resin. The flask was placed in a shaking incubator
at 20 °C and 120 rpm for 6 h. Adsorption rate was calculated
according to the following equation: adsorption rate
(%) = [(A0 A1)/A0]  100%. Where A0 is the absorbance of the pig-
ment solution before adsorption, A1 is the absorbance of the pig-
ment solution after adsorption. The absorbance was read at
475 nm.
The macroporous resin which adsorbing pigments was first
washed by deionized water and then desorbed with 20 ml of 95%
ethanol in a 100 ml conical flask. The flask was placed in a shaking
incubator at 20 °C and 120 rpm for 6 h. Desorption rate was calcu-
lated according to the following equation: desorption rate
(%) = [A2/(A0 A1)]  100%. Where A0 is the absorbance of the pig-
ment solution before adsorption, A1 is the absorbance of the pig-
ment solution after adsorption, A2 is the absorbance of the
pigment solution after desorption. The absorbance was read at
475 nm.
The sample processed by NKA-9 macroporous resin was con-
centrated firstly in a rotary vacuum evaporator at 45 °C, and then
freeze-dried to get the pigment extract for further study.
2.4. UPLC Q-TOFMS analysis
The LC–MS used for this study was an ACQUITYTM UPLC Q/TOF
PremierTM (Waters, Milford, MA, USA) equipped with an electro-
spray ionisation ion source (ESI). High purity nitrogen was used
as the nebulizer and auxiliary gas; argon was used as the collision
gas. The mass spectrometer was operated in negative ion mode
with a capillary voltage of 2.5 kV, sampling cone voltage of 35 V,
cone gas flow of 50 l/hr, desolvation gas flow of 800 l/hr, desolva-
tion temperature of 350 °C, source temperature of 120 °C, and col-
lision energy of 5.0 V. Mass spectra were collected at a rate of
1 spectra/s and the inter-scan delay was 0.02 s. Mass accuracy
was maintained by using a lock spray with leucine enkephalin
(m/z 554.2615, concentration: 2 ng/ll, flow rate: 5 ll/min) as ref-
erence. The Q-TOF instrument was operated in full scan-survey
mode. The full scan spectra from 50 to 1000 Da were acquired.
The analytical column was an ACQUITY UPLCTM BEHC18 column
(100 mm  2.1 mm, 1.7 lm, Waters, Milford, MA, USA). The two
solvents were phase A: water/formic acid (v/v, 100/0.1), phase B:
acetonitrile. The water was filtered through a 0.2 lm membrane
filter unit prior to mixing. A linear gradient was programmed: 0–
4 min: 10% B (v/v), 4–20 min: 10–70% B; 20–24 min: 70–95% B.
The flow rate was 0.3 ml/min. The column was held at 30 °C and
the injection volume was 1 ll (20 lg/ml).
2.5. DPPH radical scavenging activity
DPPH radical scavenging activity was measured according to
the method described by Chen, Wang, Rosen, and Ho (1999), with
some modifications. Briefly, 1.0 ml of sample solutions (dissolved
in 95% ethanol) with different concentrations of pigment extract
(8, 16, 31, 63, 125, and 250 lg/ml) was mixed with 2.0 ml of phos-
phate buffer (0.1 M, pH 6.0) and 2.0 ml of DPPH–ethanol solution
(200 lM). After vortex, the fluid was kept in dark at room temper-
ature for 30 min. Following centrifugation at 2000g for 10 min, the
absorbance of the supernatant was read at 517 nm. The DPPH rad-
ical scavenging activity was expressed as: scavenging rate = 1-
(As A0)/A, where As is the absorbance of the reaction solution, A0
is the absorbance of the reaction solution including 2.0 ml of etha-
nol, 2.0 ml of phosphate buffer and 1.0 ml of sample solution, and A
is the absorbance of the solution including 2.0 ml of DPPH
(200 lM), 2.0 ml of phosphate buffer and 1.0 ml of 95% ethanol.
Vitamin C was used as a positive control.
2.6. Fe2+ chelating activity
Fe2+ chelating activity was measured according to the method
described by Hsu, Coupar, and Ng (2006), with some modifications.
Briefly, the reaction mixture contained 1.0 ml of sample solution
(dissolved in 10% DMSO (v/v)) with different concentrations of pig-
ment extract (31, 63, 125, 250, 500, 750, and 1000 lg/ml), 50 ll of
FeCl2 (2 mM) and 1.0 ml of deionized water. The mixture was sha-
ken vigorously and left at room temperature for 5 min. After add-
ing 100 ll of ferrozine (5 mM), the mixture was shaken
vigorously and left for another 5 min to complex the residual
Fe2+. Following centrifugation at 2000g for 10 min, the absorbance
of the supernatant was read at 562 nm. The chelating activity was
calculated as: chelating rate = 1 (As A0)/A, where As is the absor-
 D.-Y. Zhou et al. / Food Chemistry 129 (2011) 1591–1597
bance of the reaction mixture, A0 is the absorbance of the reaction
mixture with ferrozine replaced by equivalent volume of deionized
water, and A is the absorbance of the reaction mixture with sample
replaced by equivalent volume of 10% DMSO. Ethylenediaminetet-
raacetic acid disodium salt (Na2EDTA) was used as a positive
control.
2.7. Reducing power
Reducing power was measured according to the procedure de-
scribed by Zhu, Chen, Tang, and Xiong (2008), with some modifica-
tions. One millilitre of sample solution (dissolved in 95% ethanol)
with different concentrations of pigment extract (16, 31, 63, 125,
250, and 500 lg/ml) was mixed with 1.0 ml of phosphate buffer
(0.2 M, pH 6.6) and 2.0 ml of potassium ferricyanide (1%, m/v). Fol-
lowing incubation at 50 °C for 20 min, 1.0 ml of trichloroacetic acid
(TCA, 10%, m/v) was added to the mixture. After vortex, the fluid
was centrifuged at 2000g for 10 min. Two millilitres of the upper
layer of solution were collected and mixed with 2.5 ml of deionized
water and 0.3 ml of FeCl3 (0.1%, m/v). After incubating at room
temperature for 10 min, the absorbance of the mixture was read
at 700 nm. Higher absorbance indicated greater reductive poten-
tial. Vitamin C was used as a positive control.
2.8. Inhibition of lipid peroxidation in rat liver homogenate
Lipid peroxidation inhibition activity assay was performed
according to the method described by Ren et al. (2008) and Wang,
Gao, Zhou, Cai, and Yao (2008), with some modifications. Female
Wistar rats weighting 200–250 g were purchased from the Labora-
tory Animal Center of Dalian Medical University. All animal care
and use was conducted in accordance with the Regulations for
the Administration of Affairs Concerning Experimental Animals
set by Science and Technology Department of Liaoning Province,
China. After a fasting for 12 h, the rats were sacrificed by disloca-
tion of cervical vertebra. The liver tissue was rapidly dissected
from the abdomen and homogenated with 3 times (volume of buf-
fer/weight of sample) of pre-cooled Tris–HCl buffer (40 mM, pH
7.0). Fifty microlitres of liver homogenate were mixed with
200 ll of sample solution (dissolved in 10% DMSO) with different
concentrations of pigment extract (31, 63, 125, 250, 500, 750,
and 1000 lg/ml), 50 ll of KCl (30 mM), 50 ll of FeSO4 (0.16 mM)
and 50 ll of vitamin C (0.06 mM). Following incubation at 37 °C
for 1 h, the fluid was mixed with 0.5 ml of TCA (15%; m/v) and
0.5 ml of thiobarbituric acid (0.67%; m/v). The final solution was
boiled for 15 min, cooled in cold water for 10 min, and then centri-
fuged at 2000g for 10 min. The absorbance of the supernatant was
read at 532 nm. The lipid peroxidation inhibition activity was ex-
pressed as: inhibition activity = (A + A0 As)/A, where As is the
absorbance of the reaction solution, A is the absorbance of the solu-
tion with sample replaced by equivalent volume of 10% DMSO, A0
is the absorbance of the solution with liver homogenate replaced
by equivalent volume of Tris–HCl buffer (40 mM, pH 7.0). Butyl-
ated hydroxytoluene (BHT) was used as a positive control.
2.9. Protection against oxidative stress
t-BOOH-induced macrophages protection assay was performed
according to the method described by Li, Baviello, Vlassara, and
Mitsuhashi (1997), Kaur, Athar, and Alam (2008), and Kaur, Hamid,
Ali, Alam, and Athar (2004), with some modifications. Kun-Ming
mice weighting 18–22 g was purchased from the Laboratory Ani-
mal Center of Dalian Medical University. The peritoneal exudate
cells were collected aseptically from the Kun-Ming mice according
to the process described by Sun et al. (2010). The cells were
washed twice with Hank’s buffered salt solution (HBSS) and resus-
1593
pended in RPMI 1640 culture medium. Cell viability of P95% was
confirmed by trypan blue exclusion assay (Dawson, Dawson, Lon-
don, Bredt, & Snyder, 1991). An aliquot of 200 ll of the obtained
macrophages (1  106 cells/ml) were distributed per well into a
96-well microplate (Falcon Plastics) and allowed to adhere for
2 h at 37 °C. After 2 h of incubation, nonadherent cells were re-
moved by washing 3 times with RPMI 1640 culture medium. To
test the efficacy of the pigment extract on inhibition of oxidative
stress-induced damage, macrophages were first incubated with
different concentrations of pigment extract (1, 5, and 10 lg/ml)
for 24 h. Then macrophages were exposed to oxidative stress by
adding 80 lM of t-BOOH. Following t-BOOH exposure for 2 h,
20 ll of MTT (5 mg/ml) was added and incubated for 4 h at
37 °C. Following centrifugation at 250g for 15 min, the precipitate
(formazan) was dissolved with 150 ll of DMSO. The absorbance
was thereafter determined at 490 nm. The controls were the wells
without t-BOOH exposure. Cell viability was calculated as: cell via-
bility (%) = [As/A0]  100, where As is the absorbance of sample
well, A0 is the absorbance of control well.
2.10. Statistical methods
All the tests were conducted with three replicates. Data were pre-
sented as means ± standard deviations. Differences between means
were evaluated by Least-Significant Difference Test of Analysis of
Variance and One-Sample T Test. The statistical analysis was per-
formed by using SPSS 16.0 software (SPSS Inc. Chicago, IL, USA).
Comparisons that yielded P values <0.05 were considered
significant.
3. Results and discussion
3.1. Extraction of PHNQ pigments by macroporous resin
PHNQ pigments are packaged in calcareous skeleton in sea
urchin spines. Normally, HCl-aqueous solution is used to dissolve
the calcareous skeleton to produce the free pigments (Amarowicz
et al., 1994; Kol’tsova et al., 1978; Mischenko et al., 2005; Utkina
et al., 1976). However, we found that the dissolution with HCl-
aqueous solution generated a lot of bubbles which adversely influ-
enced the subsequent extraction. In this situation, HCl–ethanol-
aqueous solution was used in this study which could dissolve the
calcareous skeleton without generating too much bubble.
Usually, diethyl ether or chloroform was used to extract and con-
centrate the PHNQ pigments (Amarowicz et al., 1994; Kol’tsova
et al., 1978; Mischenko et al., 2005; Utkina et al., 1976). However,
the volatility and toxicity of the organic solvents mean the operation
may be harmful to the operators and the environment. In the present
study, macroporous resins were used to extract and purify the PHNQ
pigments in static adsorption mode. Adsorption and desorption ef-
fects of five types of macroporous resins on the PHNQ pigments
are presented in Fig. 1. Among the five types of macroporous resins,
NKA-9 exhibited higher adsorption rate and desorption rate, and
achieved the highest recovery rate (adsorption rate  desorption
rate) of 88.99 ± 0.73%. The differences in the adsorption and desorp-
tion capacities of different resins are due to the surface areas and
polarities of the resins (Zhang, Jiang, Gao, & Li, 2008).
In addition to the recovery rate, purification factor ((sample
weight before purification/sample weight after purifica-
tion)  recovery rate) is another indicator deserving special atten-
tion. NKA-9 macroporous resin achieved a purification factor of
260.08 ± 20.19. To compare the purification effect between the
macroporous resin method and the organic solvent method, the
PHNQ pigments was extracted with diethyl ether according to
the procedure described by Amarowicz et al. (1994). The recovery
1594 D.-Y. Zhou et al. / Food Chemistry 129 (2011) 1591–1597

showed typical UV–Vis absorption spectra of PHNQ (the absorption

A a
100%
c
d
b 1
B
80%
C 2
3
D 4
60%
40%
20%
0%
D4020 D101 NKA-9 NKA-II
Adsorption rate Desorption rate Recovery rate
Fig. 1. Adsorption and desorption effect of five types of macroporous resins on the
PHNQ pigments from Strongylocentrotus nudus spines. (Values with different lower-
case letters (a-d), upper-case letters (A-E) and numbers (1-5) are significantly
different (P < 0.05)).
rate and purification factor of the diethyl ether extraction method
was 43.62 ± 1.38% and 288.95 ± 17.81, respectively. There was no
significant difference between the purification factor of the diethyl
ether extraction method and the macroporous resin method
(P > 0.05), indicating the pigment extracts obtained by the two
methods had a close purity. However, the recovery rate of the
diethyl ether extraction method was only half of that of the macro-
porous resin method, indicating the diethyl ether extraction meth-
od lost more PHNQ pigments.
3.2. Rapid identification of PHNQ by UPLC Q-TOFMS
The structures of PHNQ can influence their bioactivity. For
example, research has indicated that echinochrome hydroxyl sub-
stituents in the 2-, 3- and 7-positions play key roles in both ferrous
ion complexing and free radical scavenging capacity (Lebedev
et al., 2005). The hydroxyl groups at the 5- and 8-positions can
form hydrogen bonds with 1,4-keto groups, which hinders hydro-
gen atom donation to reactive radicals and adversely influences the
free radical scavenging capacity (Lebedev et al., 2005). To date,
about thirty PHNQ pigments from sea urchins have been identified
(Amarowicz et al., 1994; Anderson et al., 1969; Kol’tsova et al.,
1978; Mischenko et al., 2005; Utkina et al., 1976; Yakubovskaya
et al., 2007). The distribution of those PHNQ pigments shows var-
iation in different species (Anderson et al., 1969). In this situation,
the rapid characterisation of PHNQ in a sea urchin species which
has not been studied is important and may foretell whether the
PHNQ deserve further research.
In this study, UPLC Q-TOFMS was used to characterise the PHNQ
in pigment extract from S. nudus spines. There are eight visible
peaks in UPLC chromatography when UV–Vis detector monitoring
at 475 nm (Fig. 2). The compounds corresponding to these peaks all
ab
data are listed in Table 1) (Amarowicz et al., 1994; Anderson et al.,
1969; Kol’tsova et al., 1978; Mischenko et al., 2005; Utkina et al.,
1976; Yakubovskaya et al., 2007), indicating they could be classi-
fied as such type compounds. The accurate mass of the eight PHNQ
were acquired by Q-TOFMS, from which the elemental composi-
E 5 tion could be deduced (Table 1). According to the molecular for-
mula and UV–Vis spectra, and consulting with the references
(Amarowicz et al., 1994; Anderson et al., 1969; Kol’tsova et al.,
1978; Mischenko et al., 2005; Utkina et al., 1976; Yakubovskaya
et al., 2007), compounds 1, 3, 4, 6, 7, 8 were identified tentatively
as spinochrome E, 2,7-dihydroxy-naphthazarin, spinochrome B,
AB-8
spinochrome C, spinochrome A and echinochrome A, respectively.
Based on the molecular mass, the molecular formulas for com-
pounds 2 and 5 were deduced (Table 1). They may be aminated
PHNQ. To date, only two aminated hydroxynaphthazarins from
sea urchin have been reported (Mischenko et al., 2005). The molec-
ular mass and molecular formula of compounds 2 and 5 did not
match with the two known aminated hydroxynaphthazarins.
Therefore, the two compounds may be new PHNQ.
3.3. DPPH radical scavenging activity
DPPH is one of the few stable and commercially available or-
ganic nitrogen radicals which can accept an electron or hydrogen
atom to become a stable diamagnetic molecule (Soares, Dinis, Cun-
ha, & Almeida, 1997). It has been widely used as a reagent to test
the radical-scavenging ability of various samples such as natural
or artificial antioxidants because of its ease and convenience of
use. As shown in Fig. 3a, the pigment extract was found to have
the ability of scavenging DPPH radical in a dose-dependent manner
at concentration of 8 to 125 lg/ml, thereafter the scavenging activ-
ity entered plateau. Compared with a well-known antioxidant,
vitamin C, the scavenging ability of the pigment extract was signif-
icantly weaker at low concentration but stronger at high concen-
tration. The results obtained here are agreement with those
reported previously in which PHNQ pigments from sea urchin pos-
sessed excellent DPPH radical scavenging activity (Kuwahara et al.,
2009). DPPH radical abstracts a hydrogen atom from the hydroxyl
group of PHNQ to become a stable diamagnetic structure (Soares
et al., 1997). As losing two hydrogen atoms consecutively, PHNQ
maybe become naphthosemiquinone as an intermediate product
and naphthotetraketone as the final reaction product (Lebedev,
Levitskaya, Tikhonova, & Ivanova, 2001) (Fig. 4a).
3.4. Fe2+ chelating activity
Free ferrous ion is quite sensitive to oxygen and gives rise to fer-
ric ion and superoxide, thereby generating hydrogen peroxide.
Reaction of ferrous ion with hydrogen peroxide generates hydroxyl
radical, the most reactive and detrimental reactive oxygen species
(ROS) in biological systems. In this process, known as the Fenton

YUAN YANG
ZDY_090925__4 4 2: Diode Array
6.48 475
Range: 2.089e-2
1
1.5e-2 2.63

7
1.0e-2
AU

5 10.97
8.52 8
6 11.13
5.0e-3 2 3 9.91
4.58 6.15
0.97
0.0 Time
2.00 4.00 6.00 8.00 10.00 12.00 14.00

Fig. 2. UPLC separation of the pigment extract obtained from Strongylocentrotus nudus spines.
 D.-Y. Zhou et al. / Food Chemistry 129 (2011) 1591–1597 1595

Table 1
1
List of the RT, MS data of pseudomolecular ions ([M H] ), UV–Vis absorption data and structural elucidation for PHNQ identified from Strongylocentrotus nudus spines by using
UPLC/Q-TOFMS.

No. RT Mma Mcb (Da) Error Error Molecular k max k max Structural elucidation
(min) (Da) (mDa) (ppm) formula (nm)c (nm)d
1 2.63 252.9986 252.9984 0.2 0.8 C10H6O8 264, 347, 476 270, 359, 477 2,3,5,6,7,8-hexahydroxy-1,4-naphthoquinone
(Spinochrome E)
2 4.58 252.0164 252.0144 2.0 7.9 C10H7NO7 272, 370, 484 Aminopentahydroxynaphthoquinone
3 6.15 221.0106 221.0086 2.0 9.0 C10H6O6 268, 305, 484 272, 319, 486 2,5,7,8-tetrahydroxy-1,4-naphthoquinone (2,7-
dihydroxynaphthazarin)
4 6.48 221.0058 221.0086 2.8 12.7 C10H6O6 267, 318, 391, 272, 323, 385, 480 2,3,5,7-tetrahydroxy-1,4-naphthoquinone,
470 (Spinochrome B)
5 8.52 262.0352 262.0352 0 0 C10H9NO6 245, 323, 484 Acetylaminotrihydroxynaphthoquinone
6 9.91 279.0140 279.0141 0.1 0.4 C12H8O8 246, 295, 463 240, 285, 463 3-acetyl-2,5,6,7,8-pentahydroxy-1,4-naphthoquinone,
(Spinochrome C)
7 10.97 263.0182 263.0192 1.0 3.8 C12H8O7 252, 312, 485 251, 317, 520 3-acetyl-2,5,7,8-tetrahydroxy-1,4-naphthoquinone,
(Spinochrome A)
8 11.13 265.0360 265.0348 1.2 4.5 C12H10O7 254, 332, 485 260, 343, 470, 490, 6-ethyl-2,3,5,7,8-tetrahydroxy-1,4-naphthoquinone,
530 (Echinochrome A)
a
Mm, mass measured.
b
Mc, mass calculated.
c
UV–Vis absorption data measured.
d
UV–Vis absorption data reported (Anderson et al., 1969).

a b
Scavenging rate (%)

Chelating rate (%)

Vitamin C Pigments

Na2EDTA Pigments

Concentration (µg/ml) Concentration (µg/ml)

c d

Vitamin C Pigments BHT Pigments

Concentration (µg/ml) Concentration (µg/ml)

Fig. 3. Antioxidant activities of the pigment extract obtained from Strongylocentrotus nudus spines.

reaction, hydroxyl radical production is directly related to the con- ability, increasing with concentration increase. Though the chelat-
centration of ferrous ion (Galey, 1996). Chelators can form com- ing potential of the extract was weaker than that of Na2EDTA, a
plexes with metal ions and inhibit the Fenton-induced oxidation. representative metal ion chelator, which was comparable to that
As shown in Fig. 3b, the pigment extract showed obvious chelating of a black tea extract containing quinonoid pigments (Hsu et al.,
1596 D.-Y. Zhou et al. / Food Chemistry 129 (2011) 1591–1597

OH O OH O

HO OH HO O

HO OH HO O

OH O OH O

a c 2Fe3+
2DPPH Fe2+
b

2DPPH 2Fe2+
OH O OH O

HO O HO O

Fe 2+

HO O HO O

OH O OH O

OH O

HO O

HO O

OH O

Fig. 4. Mechanisms of the antioxidant activities of PHNQ. (take spinochrome E as an example).

antioxidant activity (Moridani, Pourahmad, Bui, Siraki, & O’Brien,

2003). As shown in Fig. 3c, the reducing power of the pigment ex-
tract exhibited a dose-dependent effect at a concentration range of
16 to 500 lg/ml. At a low concentration, the reducing power of the
pigment extract was lower than that of vitamin C. Whereas it was
comparable to that of vitamin C at concentrations above 500 lg/
ml. Polyphenols are good electron-donors (Moridani, Pourahmad,
Bui, Siraki, & O’Brien, 2003). In anion form, PHNQ may transmit
two electrons consecutively to ferric ion and become naphthotet-
raketone as the final reaction products (Fig. 4c).

3.6. Inhibition of lipid peroxidation in rat liver homogenate

In biological systems, ROS abstract a hydrogen atom from a

Control
Pigments (µg/ml)
Fig. 5. Protection of t-BOOH induced cytotoxicity in macrophages by the pigment
extract obtained from Strongylocentrotus nudus spines. (Values with different letters
are significantly different (P < 0.05)).
2006). Due to weak acid properties, PHNQ with ortho-hydroxyl
group are present in their divalent anion form at neutral and alkali
conditions which can form chelates with the ferrous ion (Fig. 4b)
(Lebedev et al., 2001; Lebedev et al., 2005; Sugihara, Arakawa,
Ohnishi, & Furuno, 1999). It is noteworthy that the PHNQ-metal
complexes maybe be more effective than the parent compounds
in scavenging some radicals (Sugihara et al., 1999).
3.5. Reducing power
methylene group of an unsaturated fatty acid and subsequently
form free radicals such as peroxyl radical (Matsuo, 1985). Once
these free radicals are formed, they will decompose into various
secondary oxidation products such as malondialdehyde (MDA).
Some of the breakdown products have been used as a convenient
index for determining the extent of lipid peroxidation (Matsuo,
1985; Ng, Liu, & Wang, 2000). In this study, the potential of the pig-
ment extract to inhibit lipid peroxidation was measured in rat liver
homogenate which induced by Fe2+-vitamin C system. Obviously,
the pigment extract showed an excellent inhibitory activity
(Fig. 3d). Though the activity was weaker than that of BHT at low
concentrations, it was higher than that of BHT at concentrations
above 250 lg/ml. It is postulated that the inhibition ability of
PHNQ is the result of a combination of iron chelation and free-rad-
ical scavenging activity (Lebedev et al., 2005). In contrast, BHT can
inhibit lipid peroxidation by scavenging radicals only.

Reductants can quench free radicals by transmitting electrons 3.7. Protection against oxidative stress
to them (Moridani, Pourahmad, Bui, Siraki, & O’Brien, 2003). The
electron-donating potential of a given compound, termed as reduc- Antioxidant activity of the pigment extract was also evaluated
ing capacity, may serve as a significant indicator of its potential in a cellular system. t-BOOH is an organic hydroperoxide and is
 D.-Y. Zhou et al. / Food Chemistry 129 (2011) 1591–1597
widely used to induce oxidative stress in a variety of cells (Kim
et al., 2008; Robison, Zhou, & Forman, 1995). Exposure of rat peri-
toneal macrophages to t-BOOH can lead to a considerable oxidative
stress in them, the resulting damage can cause cell death which
can either be necrotic or apoptotic (Chandra, Samali, & Orrenius,
2000). Results showed 2 h exposure to t-BOOH kills as much as
42% of macrophages (Fig. 5). However, incubation with 5 lg/ml
or more of the pigment extract before t-BOOH treatment signifi-
cantly improved the survival of macrophages from the oxidative
stress caused by t-BOOH (Fig. 5). Polyphenols such as flavonoid
can inhibit t-BOOH-induced cytotoxicity by scavenging ROS and
preventing lipid peroxidation (Dipti et al., 2006). This can explain
the protection mechanism of PHNQ as they have similar polyphe-
nolic structures.
4. Conclusion
PHNQ pigment extract was prepared from purple sea urchin (S.
nudus) spines and eight PHNQ were present in the extract. The pig-
ment extract was found to have DPPH scavenging, Fe2+ chelating,
reducing, lipid peroxidation inhibition and t-BOOH-induced mac-
rophages protection capacities. The antioxidant ability of PHNQ is
thought to be the result of a combination of iron chelation, reduc-
ing power and free-radical scavenging activity. In contrast with the
positive controls such as vitamin C, Na2EDTA and BHT, the pigment
extract exhibited lower antioxidant potent at low concentration
but close or stronger antioxidant potent at high concentration. This
does not mean the antioxidant ability of PHNQ is weaker than that
of the controls. In our opinion, the non-PHNQ compounds in the
pigment extract dilute the antioxidant activity.
Acknowledgements
This work was financially supported by ‘‘The National Great
Project of Scientific and Technical Supporting Programs Funded
by Ministry of Science & Technology of China During the 11th
Five-year Plan (No. 2008BAD94B07)’’ and ‘‘The Research Start-up
Project for Doctor Funded by Liaoning Science and Technology
Department (No. 20091002)’’.
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