Extraction and antioxidant property of polyhydroxylated naphthoquinone pigments from spines of purple sea urchin Strongylocentrotus nudus

Food Chemistry 129 (2011) 1591–1597

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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

Article history: Received 22 May 2010 Received in revised form 21 May 2011 Accepted 13 June 2011 Available online 16 June 2011 Keywords: Sea urchin Natural pigment Naphthoquinone Extraction Antioxidant

a b s t r a c t Purple sea urchin (Strongylocentrotus nudus) spines were dissolved in HCl–ethanol-aqueous solution. The polyhydroxylated 1,4-naphthoquinone (PHNQ) pigments were condensed and purified by using macroporous resin in static adsorption mode. PHNQ in the extract were characterised rapidly by using an ultra-performance liquid chromatography (UPLC) coupled to hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer (Q-TOFMS). Six known compounds including spinochrome E, 2,7dihydroxynaphthazarin, spinochrome B, spinochrome C, spinochrome A and echinochrome A, and two new compounds with molecular formula of aminopentahydroxynaphthoquinone and acetylaminotrihydroxynaphthoquinone were identified tentatively. The pigment extract was evaluated for antioxidant activity by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging capacity assay, Fe2+ chelating assay, reducing power assay, lipid peroxidation inhibition assay and tertiary-butyl hydroperoxide (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 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 distinctive 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 (Kuwahara et al., 2009). In 1938, Lederer and Glaser identified the first quinonoid 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, Denisenko, & Maksimov, 1978; Mischenko et al., 2005; Utkina, Shchedrin, & Maksimov, 1976; Yakubovskaya, Pokhilo, Mishchenko, & Anufriev, 2007). Those quinonoid pigments almost all have the ⇑ Corresponding author. Fax: +86 0411 86323262. E-mail address: [email protected] (B.-W. Zhu). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.06.014

polyhydroxylated 1,4-naphoquinone (PHNQ) structure with ethyl, 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 recent 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 ‘‘histochrome’’ 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 dissolved 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 volatile, indicating the operation is harmful to the operators and the environment. Therefore, a green and safe extraction method is necessary. 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

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for the antioxidant activity of PHNQ (Lebedev et al., 2005). Meanwhile, study indicated the predominant PHNQ are different in different 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 macroporous adsorption resin and identified tentatively by using UPLC/ Q-TOFMS. Meanwhile, the antioxidant ability of the pigment extract was tested by using DPPH scavenging capacity assay, Fe2+ chelating assay, reducing power assay, lipid peroxidation inhibition 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-diphenyltetrazolium bromide (MTT) was purchased from Ameresco Co. (Solon, 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, NKA9, NKA-II and AB-8 were used in this study. The macroporous resins were pretreated according to the manufacturer’s instructions. The adsorption and desorption procedure was performed as described 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 pigment solution before adsorption, A1 is the absorbance of the pigment 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 calculated 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 pigment 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 concentrated 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 electrospray 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, desolvation temperature of 350 °C, source temperature of 120 °C, and collision 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 reference. 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 phosphate 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 temperature for 30 min. Following centrifugation at 2000g for 10 min, the absorbance of the supernatant was read at 517 nm. The DPPH radical 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 ethanol, 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 pigment 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 shaken vigorously and left at room temperature for 5 min. After adding 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. Ethylenediaminetetraacetic acid disodium salt (Na2EDTA) was used as a positive control. 2.7. Reducing power Reducing power was measured according to the procedure described by Zhu, Chen, Tang, and Xiong (2008), with some modifications. 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). Following 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 potential. 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 Laboratory 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 dislocation of cervical vertebra. The liver tissue was rapidly dissected from the abdomen and homogenated with 3 times (volume of buffer/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 centrifuged at 2000g for 10 min. The absorbance of the supernatant was read at 532 nm. The lipid peroxidation inhibition activity was expressed as: inhibition activity = (A + A0 As)/A, where As is the absorbance of the reaction solution, A is the absorbance of the solution 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). Butylated 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 Animal 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-

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pended in RPMI 1640 culture medium. Cell viability of P95% was confirmed by trypan blue exclusion assay (Dawson, Dawson, London, 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 removed 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 viability (%) = [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 presented 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 performed 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 HClaqueous solution generated a lot of bubbles which adversely influenced the subsequent extraction. In this situation, HCl–ethanolaqueous 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 concentrate 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 effects 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 desorption 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 purification)  recovery rate) is another indicator deserving special attention. 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

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100% 80%

D.-Y. Zhou et al. / Food Chemistry 129 (2011) 1591–1597

c

b

d

A

a

showed typical UV–Vis absorption spectra of PHNQ (the absorption 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 classified as such type compounds. The accurate mass of the eight PHNQ were acquired by Q-TOFMS, from which the elemental composition could be deduced (Table 1). According to the molecular formula 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, spinochrome C, spinochrome A and echinochrome A, respectively. Based on the molecular mass, the molecular formulas for compounds 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 molecular 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.

ab

1

B C

2

3

D

4

60%

E

5

40% 20% 0% D4020

D101

NKA-9

Adsorption rate

NKA-II

Desorption rate

AB-8

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 lowercase 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 macroporous resin method, indicating the diethyl ether extraction method lost more PHNQ pigments.

3.3. DPPH radical scavenging activity DPPH is one of the few stable and commercially available organic nitrogen radicals which can accept an electron or hydrogen atom to become a stable diamagnetic molecule (Soares, Dinis, Cunha, & 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 activity entered plateau. Compared with a well-known antioxidant, vitamin C, the scavenging ability of the pigment extract was significantly weaker at low concentration but stronger at high concentration. The results obtained here are agreement with those reported previously in which PHNQ pigments from sea urchin possessed 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).

3.2. Rapid identification of PHNQ by UPLC Q-TOFMS The structures of PHNQ can influence their bioactivity. For example, research has indicated that echinochrome hydroxyl substituents 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 hydrogen 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 variation 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

3.4. Fe2+ chelating activity Free ferrous ion is quite sensitive to oxygen and gives rise to ferric 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 4

ZDY_090925__4

2: Diode Array 475 Range: 2.089e-2

6.48

1

AU

1.5e-2

2.63

7

1.0e-2

5

10.97

8

8.52

5.0e-3

2 4.58

0.97

6 3

11.13

9.91

6.15

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.

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D.-Y. Zhou et al. / Food Chemistry 129 (2011) 1591–1597 Table 1 List of the RT, MS data of pseudomolecular ions ([M H] UPLC/Q-TOFMS. No.

c d

), UV–Vis absorption data and structural elucidation for PHNQ identified from Strongylocentrotus nudus spines by using

Mma (Da)

Mcb (Da)

Error (mDa)

Error (ppm)

Molecular formula

k max (nm)c

k max (nm)d

Structural elucidation 2,3,5,6,7,8-hexahydroxy-1,4-naphthoquinone (Spinochrome E) Aminopentahydroxynaphthoquinone 2,5,7,8-tetrahydroxy-1,4-naphthoquinone (2,7dihydroxynaphthazarin) 2,3,5,7-tetrahydroxy-1,4-naphthoquinone, (Spinochrome B) Acetylaminotrihydroxynaphthoquinone 3-acetyl-2,5,6,7,8-pentahydroxy-1,4-naphthoquinone, (Spinochrome C) 3-acetyl-2,5,7,8-tetrahydroxy-1,4-naphthoquinone, (Spinochrome A) 6-ethyl-2,3,5,7,8-tetrahydroxy-1,4-naphthoquinone, (Echinochrome A)

1

2.63

252.9986

252.9984

0.2

0.8

C10H6O8

264, 347, 476

270, 359, 477

2 3

4.58 6.15

252.0164 221.0106

252.0144 221.0086

2.0 2.0

7.9 9.0

C10H7NO7 C10H6O6

272, 370, 484 268, 305, 484

272, 319, 486

4

6.48

221.0058

221.0086

2.8

12.7

C10H6O6

272, 323, 385, 480

5 6

8.52 9.91

262.0352 279.0140

262.0352 279.0141

0 0.1

0 0.4

C10H9NO6 C12H8O8

267, 318, 391, 470 245, 323, 484 246, 295, 463

7

10.97

263.0182

263.0192

1.0

3.8

C12H8O7

252, 312, 485

251, 317, 520

8

11.13

265.0360

265.0348

1.2

4.5

C12H10O7

254, 332, 485

260, 343, 470, 490, 530

240, 285, 463

Mm, mass measured. Mc, mass calculated. UV–Vis absorption data measured. UV–Vis absorption data reported (Anderson et al., 1969).

b

Scavenging rate (%)

a

Vitamin C

Pigments

Chelating rate (%)

a b

RT (min)

1

Na2EDTA

Concentration (µg/ml)

Pigments

Concentration (µg/ml)

c

d

Vitamin C

Pigments

Concentration (µg/ml)

BHT

Pigments

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 concentration of ferrous ion (Galey, 1996). Chelators can form complexes with metal ions and inhibit the Fenton-induced oxidation. As shown in Fig. 3b, the pigment extract showed obvious chelating

ability, increasing with concentration increase. Though the chelating potential of the extract was weaker than that of Na2EDTA, a representative metal ion chelator, which was comparable to that of a black tea extract containing quinonoid pigments (Hsu et al.,

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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

a

c

2DPPH

O

2Fe3+

Fe2+

b 2Fe2+

2DPPH OH

OH

O

HO

O

O

HO

O Fe 2+

HO

O OH

O

OH

O

HO

HO

O OH

O

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 extract 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 naphthotetraketone as the final reaction products (Fig. 4c). 3.6. Inhibition of lipid peroxidation in rat liver homogenate

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 Reductants can quench free radicals by transmitting electrons to them (Moridani, Pourahmad, Bui, Siraki, & O’Brien, 2003). The electron-donating potential of a given compound, termed as reducing capacity, may serve as a significant indicator of its potential

In biological systems, ROS abstract a hydrogen atom from a 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 pigment 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-radical scavenging activity (Lebedev et al., 2005). In contrast, BHT can inhibit lipid peroxidation by scavenging radicals only. 3.7. Protection against oxidative stress Antioxidant activity of the pigment extract was also evaluated in a cellular system. t-BOOH is an organic hydroperoxide and is

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widely used to induce oxidative stress in a variety of cells (Kim et al., 2008; Robison, Zhou, & Forman, 1995). Exposure of rat peritoneal 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 significantly 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 polyphenolic structures. 4. Conclusion PHNQ pigment extract was prepared from purple sea urchin (S. nudus) spines and eight PHNQ were present in the extract. The pigment extract was found to have DPPH scavenging, Fe2+ chelating, reducing, lipid peroxidation inhibition and t-BOOH-induced macrophages protection capacities. The antioxidant ability of PHNQ is thought to be the result of a combination of iron chelation, reducing 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)’’. References Amarowicz, R., Synowiecki, J., & Shahidi, F. (1994). Sephadex LH-20 separation of pigments from shells of red sea urchin (Strongylocentrotus franciscanus). Food Chemistry, 51, 227–229. Anderson, H. A., Mathieson, J. W., & Thomson, R. H. (1969). Distribution of spinochrome pigments in echinoids. Comparative Biochemistry and Physiology, 28, 333–345. Chandra, J., Samali, A., & Orrenius, S. (2000). Triggering and modulation of apoptosis by oxidative stress. Free Radical Biology and Medicine, 29, 323–333. Chen, Y., Wang, M. F., Rosen, R. T., & Ho, C. T. (1999). 2,2-diphenyl-1-picrylhydrazyl radical-scavenging active components from Polygonum multiflorum Thunb. Journal of Agricultural and Food Chemistry, 47, 2226–2228. Dawson, V. L., Dawson, T. M., London, E. D., Bredt, D. S., & Snyder, S. H. (1991). Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures. Proceedings of the National Academy of Sciences of the United States of America, 88, 6368–6371. De la Cruz-Garcia, C., Lopez-Hernandez, J., Gonzalez-Castro, M. J., De Quiros, A. R. B., & Simal-Lozano, J. (2000). Protein, amino acid and fatty acid contents in raw and canned sea urchin (Paracentrotus lividus) harvested in Galicia (NW Spain). Journal of the Science of Food and Agriculture, 80, 1189–1192. Dipti, P., Sharma, S. K., Sairam, M., Ilavazhagan, G., Sawhney, R. C., & Banerjee, P. K. (2006). Flavonoids protect U-937 macrophages against tert-butylhydroperoxide induced oxidative injury. Food and Chemical Toxicology, 44, 1024–1030. Galey, J. B. (1996). Potential use of iron chelators against oxidative damage. Advances in Pharmacology, 38, 167–203.

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