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Antioxidant activity of sulfated polysaccharide fractions extracted from Undaria pinnitafida in vitro
International Journal of Biological Macromolecules 46 (2010) 193–198
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Antioxidant activity of sulfated polysaccharide fractions extracted from Undaria pinnitafida in vitro Tingting Hu, Dan Liu, Yan Chen, Jun Wu, Shusheng Wang ∗ School of Life Sciences, Anhui University, Feixi Street, Hefei 230039, PR China
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Article history: Received 10 October 2009 Received in revised form 20 November 2009 Accepted 9 December 2009 Available online 16 December 2009 Keywords: Sulfated polysaccharide Antioxidant activity Radical-scavenging effect Chelating ability Undaria pinnitafida
a b s t r a c t Two sulfated polysaccharide fractions (S1 and S2) were successfully isolated from seaweed Undaia pinnitafida and the chemical characteristics were determined. Antioxidant activities of the polysaccharide fractions were evaluated by assays of various antioxidants in vitro systems, including superoxide anion, 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl radical-scavenging activity and metal chelating ability. The results showed that the two sulfated polysaccharides contained rhamnose as the major neutral sugar and present high sulfate content (33.99–34.29%). Antioxidant assays suggested that the two sulfated polysaccharide fractions (S1 and S2) possessed good antioxidant properties and had stronger antioxidant abilities than de-sulfated polysaccharides (DS-1 and DS-2). Available data obtained by in vitro models suggested that the correlation between the sulfate content and antioxidant activity was positive. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Reactive oxygen species (ROS) are generated by normal metabolic process or from exogenous factors and agents. These reactive oxygen species and free radical-initiated reactions are known to induce a wide variety of pathological effects, such as carcinogenesis, atherosclerosis and DNA damage as well as in degenerative processes associated with aging [1–3]. Antioxidants are substances that delay or prevent the oxidation of cellular oxidizable substrates. They work in two ways: scavenging ROS and inhibiting the generation of ROS. The most commonly used antioxidants at the present time are butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG) and tertbutylhydroxytoluene (TBHQ). However, most of the antioxidants used have been suspected of being responsible for liver damage and carcinogenesis [4]. Thus, it is essential to develop and utilize effective natural antioxidants so that they can protect the human body from free radicals and retard the progress of many chronic diseases [5]. Recently, algal polysaccharides have been demonstrated to play an important role as free radical scavengers in vitro and antioxidants for the prevention of oxidative damage in living organisms [6]. The brown alga, Undaria pinnitafida, is an important food source in many parts of the world. U. pinnitafida is nutritious, having abun-
dant protein, polysaccharide, and minerals. Besides being widely utilized as food, it has been used in traditional Chinese medicine as a drug for over 1000 years. Polysaccharide extracted from U. pinnitafida is a group of sulfated heteropolysaccharide. Sulfated polysaccharide from marine algae contains diverse biological activities in potential medicinal value, such as anticoagulant, antitumor, antiviral and antioxidant [7–13]. Their activity depends on several structural parameters such as the degree of sulfation (DS), the molecular weight and type of sugar [14]. Schaeffer and Krylov [15] found that the sulfate component of fucoidan is related to the beneficial effects of the natural compound on anti-HIVA infection. Koyanagi et al. [8] reported the oversulfation of fucoidan significantly increases its antitumor and anti-angiogenic effects. These reports suggest that the ester sulfate of the polysaccharide may be an active component responsible for these biological activities. In this study, two polysaccharide fractions were isolated from U. pinnitafida. Their antioxidant activities in vitro were investigated, including superoxide, hydroxyl, DPPH radicals-scavenging effects and chelating ability. Their chemical characteristics and antioxidant properties were reported in this paper. 2. Materials and methods 2.1. Chemicals
∗ Corresponding author. Tel.: +86 13866778630. E-mail address: [email protected] (S. Wang). 0141-8130/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2009.12.004
DEAE-Sephadex A-25 and Superdex-200 were purchased from Amersham Biosciences Co. 2,2-Diphenyl-1-picrylhydrazyl (DPPH)
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was from Sigma Chemicals Co. Dialysis membranes were produced by Spectrum Co., and molecular weight was cut off at 3500 Da. Standard monosaccharides (rhamnose, fucose, arabinose, xylose, mannose, galactose, glucose, glucuronic acid, and galacturonic acid) were purchased from Sigma–Aldrich. All other reagents used were of analytical grade. U. pinnitafida was collected on the coast of Qingdao, China. Algae were washed, air dried and kept in plastic bags at room temperature before using.
2.2. Extraction of the crude sulfated polysaccharides from U. pinnitafida Dried algae were extracted in 0.4% 12 l HCl (pH 2) for 24 h. The solution was separated from algae residues by successive filtration through gauze, and the residues were extracted with 10 l of HCl (0.4%) at 60 ◦ C for 6 h. All extracts were combined, neutralized with NaOH, concentrated using a evaporator at 65 ◦ C under reduced pressure. The proteins in the product of condensation were removed using the Sevage reagent three times. After the removal of the Sevage reagent, the extract was precipitated with threefold of the volume of 95% ethanol (v/v) and kept at 4 ◦ C overnight. The precipitate was collected by centrifugation, and washed three times with anhydrous ethanol. It was then dissolved in distilled water and dialyzed against distilled water at room temperature for two successive days. The retained fraction was recovered, concen-
trated under reduced pressure and lyophilized to obtain the crude polysaccharide.
2.3. Further purification The crude polysaccharide (100 mg) was dissolved in 0.9% NaCl and fractionated by a DEAE-Sephadex A-25 column (2.5 cm × 60 cm) eluting with a step gradient of 0.1–2 M NaCl. Total sugar content of the elution was determined by the phenol–sulfuric acid method. The elution profile detected by the phenol–sulfuric acid assay showed two big elution peaks namely S1 and S2, respectively. To test the homogeneity of S1 and S2, the fractions were further applied to a Hiload 16/60 Superdex-200 (1.6 cm × 60 cm) with the AKTA Purifier system and eluted with 0.9% NaCl at a flow rate of 1 ml/min.
2.4. Chemical analysis Total carbohydrate content was estimated by the phenol–sulfuric acid method using rhamnose as standard [16]. Protein content was measured by the method of Bradford [17]. Sulfate content was analyzed with the method of Hartiala and Therho [18]. Uronic acid was estimated in a modified carbazole method using d-glucuronic acid as standard [19].
Fig. 1. (A) Purification of the polysaccharides obtained from seaweed Undaia pinnitafida. The crude polysaccharide was applied to a column of DEAE-Sephadex A-25 and eluted as described in Section 2. The fractions containing the polysaccharides were pooled and named as S1 and S2, respectively. (B) S1 obtained on DEAE-Sephadex A-25 was applied to a Superdex-200 and eluted as described in Section 2. (C) S2 obtained on DEAE-Sephadex A-25 was applied to a Superdex-200 and eluted as described in Section 2.
T. Hu et al. / International Journal of Biological Macromolecules 46 (2010) 193–198 Table 1 Chemical component of S1, S2, DS-1 and DS-2. Component
Carbohydrate (%)
Uronic acid (%)
Sulfate (%)
Protein (%)
S1 S2 DS-1 DS-2
40.54 33.50 75.71 72.32
12.97 9.98 9.95 9.91
34.29 33.99 6.09 5.85
1.39 1.41 1.11 1.14
2.5. Monosaccharide composition analysis Purified polysaccharide (5 mg) was hydrolyzed using anhydrous methanol containing 2 M HCl at 80 ◦ C for 20 h. Then the hydrolyzed products were neutralized with methanol–KOH and dried. The above dried product was dissolved with 0.2 ml pyridine adding 3 mg mannitol (as internal references) at 75 ◦ C for 30 min. After cooling to room temperature, hexamethyldisilazane (0.2 ml) and trimethylchlorosilane (0.1 ml) were added to the mixture. Then the trimethylsilylated derivatives were analyzed by GC (HP2010) using a RTX-WAX column and a flame ionization detector. The operation was performed using the following conditions—N2 : 0.8 ml/min; H2 : 1.5 ml/min; air: 200 ml/min; injection temperature: 200 ◦ C; detector temperature: 200 ◦ C. Sugar identification was done by comparison with reference sugars (rhamnose, fucose, arabinose, xylose, mannose, galactose, glucose, glucuronic acid, and galacturonic acid). 2.6. IR spectroscopy analysis For IR spectroscopy, the polysaccharide was mixed with KBr powder, ground and then pressed into a 1 mm pellets for Fourier transform infrared (FI-IR) measurement in the frequency range of 4000–500 cm−1 . FI-IR spectra of the polysaccharides were measured on a Nicolet Nexus870 spectrometer.
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buffer (150 mM, PH 7.4) for 30 min at 37 ◦ C, and hydroxyl radical was detected by monitoring absorbance at 520 nm. In the control, sample was substituted with distilled water, and sodium phosphate buffer replaced H2 O2 . The capability of scavenging hydroxyl radical was calculated using the following equation: scavenging effect (%) = [1 − A1 /A0 ] × 100, where A0 is the absorbance of the control and A1 is the absorbance of the sample. 2.8.3. Scavenging ability on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals The scavenging activity of the DPPH free radical was assayed according to the method of Kargozler et al. [23] with slight modification. In brief, 2 ml of sample solution at different concentrations (0.2–1.4 mg/ml) was added to 2 ml 0.2 mM ethanol solution of DPPH, the reaction mixture was shaken vigorously and incubated for 30 min in the dark at room temperature. The absorbance of the resulting solution was measured at 517 nm. The ability of scavenging the DPPH radicals was calculated using the following equation: scavenging effect (%) = [(A0 − A1 )/A0 ] × 100, where A0 is the absorbance of DPPH solution without the tested samples and A1 is the absorbance of the tested samples with DPPH solution. 2.8.4. Metal chelating assay The ferrous ion chelating effects of all samples were investigated according to the method reported by Liu et al. [24]. Briefly, the reaction mixture, containing different concentrations of samples (0.2–2.0 mg/ml), FeCl2 (0.1 ml, 2 mM), and ferroizine (0.4 ml, 5 mM), was shaken well and incubated for 10 min at room temperature. The absorbance of the mixture was measured at 562 nm against a blank. The ability of different concentrations of samples to chelate ferrous ion was calculated using the following equation: chelating ability (%) = [(A0 − A1 )/A0 ] × 100, where A0 is the absorbance of the control reaction and A1 is the absorbance of the sample. 2.9. Statistical analysis
2.7. De-sulfation Partial solvolytic de-sulfation was carried out as previously described [20]. The polysaccharides S1 and S2 in the pyridinium salt form were treated with 89:10:1 Me2 SO–MeOH–pyridine at 100 ◦ C for 8 h. The de-sulfated polysaccharides were recovered after dialysis and freeze-drying to afford DS-1 and DS-2.
All bioassay results were expressed as means ± standard deviation (SD). The experimental data were subjected to an analysis of variance (ANOVA) for a completely random design. For each polysaccharide, three samples were prepared for assays of every antioxidant attribute. 3. Results and discussion
2.8. Determination of antioxidant activity 3.1. Isolation and purification of the polysaccharide 2.8.1. Scavenging ability on superoxide radical The scavenging effect on superoxide radicals was assessed according to the method of Marklund and Marklund [21] with a minor modification. Briefly, 3 ml 0.05 mol/l Tris–HCl buffer (pH 8.2) and 1 ml of samples at different concentrations (0.05–0.21 mg/ml) were incubated at 25 ◦ C for 10 min, then 200 l of pyrogallol at the same temperature were added to the mixture, and the reaction was proceeded at 25 ◦ C for 4 min. Finally, the reaction system was terminated by the addition of 0.5 ml of HCl. The absorbance of the mixture was measured at 320 nm against the blank. The percentage inhibition of superoxide anion radicals scavenging was calculated using the following formula: scavenging effect (%) = [1 − A1 /A0 ] × 100, where A0 is the absorbance of control without the tested samples and A1 is the absorbance in the presence of the tested samples. 2.8.2. Scavenging ability on hydroxyl radical Hydroxyl radical-scavenging activity was measured by the method of Smirnoff and Cumbes [22] with a minor modification. The reaction mixture, containing different samples (0.4–1.6 mg/ml), was incubated with 2 mM EDTA–Fe (0.5 ml), 3% H2 O2 (1 ml), and 360 g/ml crocus in 4.5 ml sodium phosphate
The crude polysaccharide was extracted from U. pinnitafida and then purified by DEAE-Sephadex A-25 column, and two fractions were obtained: S1 and S2 from 1.1 and 1.6 M NaCl elution, respectively (Fig. 1A). Almost no polysaccharide was detected in 0–1 or 2.0 M NaCl elution. The two polysaccharide fractions were, respectively, pooled, dialyzed and lyophilized. The yields of the two fractions were 10.05% and 57.35% S1 and S2, respectively, appeared as only a single and symmetrically sharp peak on Superdex-200 (1.6 cm × 60 cm) with the AKTA Purifier system, indicating the two polysaccharide fractions (S1 and S2) were homogenous polysaccharides (Fig. 1B and C). 3.2. Chemical analysis The chemical composition of the polysaccharide was given in Tables 1 and 2. The results showed the main chemical components of all samples were rhamnose and sulfate, along with uronic acid and a small amount of protein. The sulfated polysaccharides (S1 and S2) had the higher sulfate content than the de-sulfated polysaccharides (DS-1 and DS-2). The rhamnose content in S1, S2,
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Table 2 Monosaccharide composition of S1, S2, DS-1 and DS-2 (mol%). Component S1 S2 DS-1 DS-2 a
Rha 51.44 40.30 50.37 39.94
Xyl 25.34 17.66 24.81 20.33
Gal 10.28 34.48 14.25 32.45
Ara
Glc
Fuc
a
a
a
nd nda nda nda
nd nda nda nda
nd nda nda nda
Man a
nd nda nda nda
GlcA
GalA
12.94 7.56 10.57 7.28
nda nda nda nda
Not detectable below the limit at 0.001.
DS-1 and DS-2 was 40.54%, 33.50%, 75.71% and 73.72%, respectively From gas chromatography, rhamnose was the main sugar unit, accounting for 51.44, 40.30, 50.37 and 39.94 mol% of the total sugar in S1, S2, DS-1 and DS-2. Xylose (24.81–25.34 mol%) was the second main sugar in the sample S1 and DS-1, while galactose (32.45–34.48 mol%) was the second main sugar in S2 and DS-2. In addition to rhamnose, xylose and galactose, glucuronic acid was also seen in the samples, which was consistent with the results of carbazole–sulfuric acid reaction. These results demonstrated that the polysaccharide (S1 and S2) isolated from U. pinnitafida had a different chemical composition to Phaeophyta from other species, was high rhamonse-containing sulfated polysaccharide. 3.3. IR spectroscopy FT-IR spectra of the four polysaccharides were similar. The signals around 3410–3420 cm−1 were from the stretch vibration of O–H existed in the hydrogen bond of the molecules. Signals at 2940 cm−1 were from the stretch vibration of –CH; IR spectrum of the sulfated polysaccharide demonstrated several bands corresponding to sulfate ester: the peaks at 847 and 1260 cm−1 derived from the bending vibration of C–O–S of the sulfate in axial position and stretching vibration of S–O of sulfate, respectively. The signals around 1260 cm−1 of DS-1 and DS-2 were weaker than S1 and S2, which means the content of sulfate group was lower than S1 and S2. In addition, signals at 1620–1635 cm−1 were attributed to the asymmetric stretch vibration of COO− of uronic acids. 1430 cm−1 was due to the symmetric stretch vibrations of COO− and the stretch vibration of C–O within –COOH. 3.4. Scavenging ability on superoxide radicals Superoxide radical can be generated by pyrogallol autooxidation and it can produce a colored compound. Resulting from a color change from purple to yellow, the absorbance at 320 nm increased when the superoxide anion was scavenged by an antioxidant, which can represent the content of superoxide radicals and indicate the antioxidant activity of the sample [25]. As shown in Fig. 2, the scavenging effects of all tested samples on superoxide radicals
Fig. 2. Scavenging effect of S1, S2, DS-1 and DS-2 on superoxide radicals. Results were representative of three separated experiments.
Fig. 3. Scavenging effect of S1, S2, DS-1 and DS-2 on hydroxyl radicals. Results were representative of three separated experiments.
were shown significantly in a concentration-dependent manner. A significant increase of the scavenging activity was observed at the concentration range (50–100 g/ml) of the polysaccharides. At a concentration of 210 g/ml, the scavenging activities were 82.65%, 80.02%, 49.93% and 48.01% for S1, S2, DS-1, and DS-2, respectively. But the scavenging effect of vitamin C was only 69.2% at a concentration of 200 g/ml. Compared to this result, the sulfated polysaccharide had stronger scavenging activity for superoxide radical than Vitamin C. Moreover, S1 and S2 had significantly higher scavenging activities on superoxide radicals than DS-1 and DS-2. This demonstrated that the sulfate content affected their antioxidant activity, which was in accordance with Zhang et al. [26] and Wang et al. [27] that higher sulfate content showed greater scavenging effect of superoxide radical. Among different reactive oxygen species (ROS), superoxide is generated first [28]. Although superoxide is a relatively weak oxidant, it may decompose to form stronger ROS, such as singlet oxygen and hydroxyl radical, which initiate peroxidation of lipids. Further, superoxide is also known to initiate indirectly the lipid peroxidation as a result of H2 O2 formation, creating precursors of hydroxyl radicals [29]. Therefore, superoxide scavenging is extremely important to antioxidant work. Our date on the activity of scavenging superoxide radicals suggested that it was likely to contribute towards the observed antioxidant effect. 3.5. Scavenging ability on hydroxyl radicals Hydroxyl radical is considered to be a highly potent oxidant which can react with most biomacromolecules functioning in living cells and induce severe damage to the adjacent biomolecules. Thus, removing hydroxyl radical is important for antioxidant defense in cell or food systems. As shown in Fig. 3, the two sulfated polysaccharides isolated from U. pinnitafida exhibited a strong scavenging activity on hydroxyl radicals. The scavenging ability of polysaccharides on hydroxyl radicals was in a concentration-dependent way. A significant increase of the scavenging effect was observed at the concentration range (0.4–1 mg/ml) of the samples. It was reported that scavenging effect on hydroxyl radicals of vitamin C was 50.0% and 60.0% at 1.63 mg/ml [30]. But the effects of S1 and S2 were 80.01% and 78.91% at 1.6 mg/ml, respectively. Moreover, all the sulfated polysaccharide fractions were found to have higher scavenging ability on hydroxyl radicals than de-sulfated samples. The present results proved that the sulfated polysaccharides isolated from U. pinnitafida were good scavengers for hydroxyl radicals and the sulfate group played an important role in the scavenging of hydroxyl radicals. Previous studies had reported that there are two type of antioxidation mechanism: one suppresses the generation of the hydroxyl
T. Hu et al. / International Journal of Biological Macromolecules 46 (2010) 193–198
Fig. 4. Scavenging effect of S1, S2, DS-1 and DS-2 on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. Results were representative of three separated experiments.
radical and the other scavenges the hydroxyl radicals generated. In the former, the antioxidant activity may ligate to the metal ions which H2 O2 to give the metal complexes. The metal complexes thus formed cannot further react with H2 O2 to give a hydroxyl radical [31,32]. In another assay system in this study, it was demonstrated that the iron chelating ability and the trend of the chelating ability were nearly similar to the order of the scavenging ability to hydroxyl radical. The antioxidant activities of the tested samples were not a function of a single factor but a combination of several factors. 3.6. Free radical-scavenging activity on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical The DPPH free radical is a stable free radical, which has been widely accepted as a tool for estimating the free radical-scavenging activities of antioxidants [33]. When DPPH free radical encounters a proton-donating substance, for example, an antioxidant, the radical would be scavenged and the absorbance at 517 nm is reduced. Based on this principle, the antioxidant activity of a substance can be expressed as its ability in scavenging the DPPH free radical [34]. As shown in Fig. 4, the scavenging abilities of all samples were in a concentration-dependent fashion. At a concentration of 0.6 mg/ml, the four polysaccharides showed scavenging abilities of 4.9–20.21% on DPPH radicals. At 1.4 mg/ml, the scavenging effect increased to 30.87%, 35.01%, 56.12% and 60.88% for S1, S2, DS-1 and DS-2, respectively. Apparently, S1 and S2 had higher scavenging abilities than DS-1 and DS-2. However, the scavenging effects of the four polysaccharides on DPPH radicals were all relatively lower than that of ascorbic acid at the same concentration. 3.7. Chelating effect on ferrous ions Fig. 5 showed ferrous ion chelating effects of S1, S2 and DS-2 were concentration related and that of DS-1 was not concentration dependent. At a concentration of 0.2 mg/ml, the chelating effect of DS-1 was stronger than that of high content polysaccharide, but it was not significant. At high concentration, the effects of S1 and S2 were more pronounced than that of de-sulfated samples, and the effect of DS-1 was the lowest. Metal chelating activity is claimed as one of antioxidant mechanisms, since it reduces the concentration of the catalyzing transition metal in lipid peroxidation. Among the transition metals, iron is known as the most important lipid oxidation prooxidant due to its high reactivity. The ferrous state of iron accelerates lipid oxidation by breaking down hydrogen and lipid peroxides to reactive free radicals via the Fenton reaction
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Fig. 5. Chelating ability of S1, S2, DS-1 and DS-2 on ferrous ions. Results were representative of three separated experiments.
(Fe2+ + H2 O2 → Fe3+ + OH− + OH• ). Fe3+ ion also produces radicals from peroxides although the rate is less than tenth that of Fe2+ ion. Thus Fe2+ ion is the most powerful prooxidant among the various species of metal ions [35]. The date of Fe2+ chelating effect demonstrated that the sulfated polysaccharides S1 and S2 exhibited moderate chelating activity. 4. Conclusion According to the results stated above, it could be concluded the crude polysaccharide extracted from U. pinnitafia predominantly contained two polysaccharide fractions (S1 and S2) after purified by DEAE-Sephadex A-25 column chromatography. The study also showed two polysaccharide fractions were high rhamnosecontaining sulfated polysaccharides. In addition, S1 and S2 were antioxidative and exhibited stronger antioxidant activities compared to that of de-sulfated polysaccharides in vitro. A positive correlation has been revealed between sulfate content and antioxidant activity. Overall, the present experiments on bioactivity of the sulfated polysaccharide from U. pinnitafia showed it was useful as functional food as well as potential therapeutic agent. Acknowledgement This work was supported by the National Natural Science Foundation of China (30800194). References [1] G. Blander, R.M. Oliveira, C.M. Conboy, M. Haigis, L. Guarente, J. Biol. Chem. 278 (2003) 38966–38969. [2] F. Liu, V.E.C. Ooi, S.T. Chang, Life Sci. 60 (1997) 763–771. [3] J.L. Mau, H.C. Lin, C.C. Chen, J. Agric. Food Chem. 50 (2002) 6072–6077. [4] H.C. Grice, Food Chem. Toxicol. 26 (1988) 717–723. [5] S. Nandita, P.S. Rajini, Food Chem. 85 (2004) 611–616. [6] Q.B. Zhang, N. Li, X.G. Liu, Z.Q. Zhao, Z.H. Xu, Carbohydr. Res. 339 (2004) 105–111. [7] T. Nishino, T. Nagumo, Carbohydr. Res. 229 (1992) 355–362. [8] S. Koyanagi, N. Tanigawa, H. Nakagawa, S. Soeda, H. Shimeno, Biochem. Pharamacol. 65 (2003) 173–179. [9] N.M.A. Ponce, C.A. Pujol, E.B. Damonte, M.L. Flores, C.A. Stoerz, Carbohydr. Res. 338 (2003) 153–165. [10] H. Qi, Q. Zhang, T. Zhao, R. Hu, K. Zhang, Z. Li, Bioorg. Med. Chem. Lett. 16 (2006) 2441–2445. [11] H.J. Zhang, W.J. Mao, F. Fang, H.Y. Li, H.H. Sun, Y. Chen, X.H. Qi, Carbohydr. Polym. 71 (2008) 428–434. [12] J.B. Lee, K. Hayashi, M. Maeda, T. Hayashi, Planta Med. 65 (1999) 439–441. [13] S.R. Sem, A.K. Das, A.K. Siddhanta, K.H. Mody, B.K. Ramavat, V.D. Chauhan, J.R. Vedasiromoni, D.K. Ganguly, Int. J. Biol. Macromol. 16 (1994) 279–280. [14] M.R.S. Melo, J.P.A. Feitosa, A.L.P. Freitas, R.C.M. de Paula, Carbohydr. Polym. 49 (2002) 491–498. [15] D.J. Schaeffer, V.S. Krylov, Ecotoxicol. Environ. Safety 45 (2000) 208–227.
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Antioxidant activity of sulfated polysaccharide fractions extracted from Undaria pinnitafida in vitro Tingting Hu, Dan Liu, Yan Chen, Jun Wu, Shusheng Wang ∗ School of Life Sciences, Anhui University, Feixi Street, Hefei 230039, PR China
a r t i c l e
i n f o
Article history: Received 10 October 2009 Received in revised form 20 November 2009 Accepted 9 December 2009 Available online 16 December 2009 Keywords: Sulfated polysaccharide Antioxidant activity Radical-scavenging effect Chelating ability Undaria pinnitafida
a b s t r a c t Two sulfated polysaccharide fractions (S1 and S2) were successfully isolated from seaweed Undaia pinnitafida and the chemical characteristics were determined. Antioxidant activities of the polysaccharide fractions were evaluated by assays of various antioxidants in vitro systems, including superoxide anion, 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl radical-scavenging activity and metal chelating ability. The results showed that the two sulfated polysaccharides contained rhamnose as the major neutral sugar and present high sulfate content (33.99–34.29%). Antioxidant assays suggested that the two sulfated polysaccharide fractions (S1 and S2) possessed good antioxidant properties and had stronger antioxidant abilities than de-sulfated polysaccharides (DS-1 and DS-2). Available data obtained by in vitro models suggested that the correlation between the sulfate content and antioxidant activity was positive. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Reactive oxygen species (ROS) are generated by normal metabolic process or from exogenous factors and agents. These reactive oxygen species and free radical-initiated reactions are known to induce a wide variety of pathological effects, such as carcinogenesis, atherosclerosis and DNA damage as well as in degenerative processes associated with aging [1–3]. Antioxidants are substances that delay or prevent the oxidation of cellular oxidizable substrates. They work in two ways: scavenging ROS and inhibiting the generation of ROS. The most commonly used antioxidants at the present time are butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG) and tertbutylhydroxytoluene (TBHQ). However, most of the antioxidants used have been suspected of being responsible for liver damage and carcinogenesis [4]. Thus, it is essential to develop and utilize effective natural antioxidants so that they can protect the human body from free radicals and retard the progress of many chronic diseases [5]. Recently, algal polysaccharides have been demonstrated to play an important role as free radical scavengers in vitro and antioxidants for the prevention of oxidative damage in living organisms [6]. The brown alga, Undaria pinnitafida, is an important food source in many parts of the world. U. pinnitafida is nutritious, having abun-
dant protein, polysaccharide, and minerals. Besides being widely utilized as food, it has been used in traditional Chinese medicine as a drug for over 1000 years. Polysaccharide extracted from U. pinnitafida is a group of sulfated heteropolysaccharide. Sulfated polysaccharide from marine algae contains diverse biological activities in potential medicinal value, such as anticoagulant, antitumor, antiviral and antioxidant [7–13]. Their activity depends on several structural parameters such as the degree of sulfation (DS), the molecular weight and type of sugar [14]. Schaeffer and Krylov [15] found that the sulfate component of fucoidan is related to the beneficial effects of the natural compound on anti-HIVA infection. Koyanagi et al. [8] reported the oversulfation of fucoidan significantly increases its antitumor and anti-angiogenic effects. These reports suggest that the ester sulfate of the polysaccharide may be an active component responsible for these biological activities. In this study, two polysaccharide fractions were isolated from U. pinnitafida. Their antioxidant activities in vitro were investigated, including superoxide, hydroxyl, DPPH radicals-scavenging effects and chelating ability. Their chemical characteristics and antioxidant properties were reported in this paper. 2. Materials and methods 2.1. Chemicals
∗ Corresponding author. Tel.: +86 13866778630. E-mail address: [email protected] (S. Wang). 0141-8130/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2009.12.004
DEAE-Sephadex A-25 and Superdex-200 were purchased from Amersham Biosciences Co. 2,2-Diphenyl-1-picrylhydrazyl (DPPH)
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was from Sigma Chemicals Co. Dialysis membranes were produced by Spectrum Co., and molecular weight was cut off at 3500 Da. Standard monosaccharides (rhamnose, fucose, arabinose, xylose, mannose, galactose, glucose, glucuronic acid, and galacturonic acid) were purchased from Sigma–Aldrich. All other reagents used were of analytical grade. U. pinnitafida was collected on the coast of Qingdao, China. Algae were washed, air dried and kept in plastic bags at room temperature before using.
2.2. Extraction of the crude sulfated polysaccharides from U. pinnitafida Dried algae were extracted in 0.4% 12 l HCl (pH 2) for 24 h. The solution was separated from algae residues by successive filtration through gauze, and the residues were extracted with 10 l of HCl (0.4%) at 60 ◦ C for 6 h. All extracts were combined, neutralized with NaOH, concentrated using a evaporator at 65 ◦ C under reduced pressure. The proteins in the product of condensation were removed using the Sevage reagent three times. After the removal of the Sevage reagent, the extract was precipitated with threefold of the volume of 95% ethanol (v/v) and kept at 4 ◦ C overnight. The precipitate was collected by centrifugation, and washed three times with anhydrous ethanol. It was then dissolved in distilled water and dialyzed against distilled water at room temperature for two successive days. The retained fraction was recovered, concen-
trated under reduced pressure and lyophilized to obtain the crude polysaccharide.
2.3. Further purification The crude polysaccharide (100 mg) was dissolved in 0.9% NaCl and fractionated by a DEAE-Sephadex A-25 column (2.5 cm × 60 cm) eluting with a step gradient of 0.1–2 M NaCl. Total sugar content of the elution was determined by the phenol–sulfuric acid method. The elution profile detected by the phenol–sulfuric acid assay showed two big elution peaks namely S1 and S2, respectively. To test the homogeneity of S1 and S2, the fractions were further applied to a Hiload 16/60 Superdex-200 (1.6 cm × 60 cm) with the AKTA Purifier system and eluted with 0.9% NaCl at a flow rate of 1 ml/min.
2.4. Chemical analysis Total carbohydrate content was estimated by the phenol–sulfuric acid method using rhamnose as standard [16]. Protein content was measured by the method of Bradford [17]. Sulfate content was analyzed with the method of Hartiala and Therho [18]. Uronic acid was estimated in a modified carbazole method using d-glucuronic acid as standard [19].
Fig. 1. (A) Purification of the polysaccharides obtained from seaweed Undaia pinnitafida. The crude polysaccharide was applied to a column of DEAE-Sephadex A-25 and eluted as described in Section 2. The fractions containing the polysaccharides were pooled and named as S1 and S2, respectively. (B) S1 obtained on DEAE-Sephadex A-25 was applied to a Superdex-200 and eluted as described in Section 2. (C) S2 obtained on DEAE-Sephadex A-25 was applied to a Superdex-200 and eluted as described in Section 2.
T. Hu et al. / International Journal of Biological Macromolecules 46 (2010) 193–198 Table 1 Chemical component of S1, S2, DS-1 and DS-2. Component
Carbohydrate (%)
Uronic acid (%)
Sulfate (%)
Protein (%)
S1 S2 DS-1 DS-2
40.54 33.50 75.71 72.32
12.97 9.98 9.95 9.91
34.29 33.99 6.09 5.85
1.39 1.41 1.11 1.14
2.5. Monosaccharide composition analysis Purified polysaccharide (5 mg) was hydrolyzed using anhydrous methanol containing 2 M HCl at 80 ◦ C for 20 h. Then the hydrolyzed products were neutralized with methanol–KOH and dried. The above dried product was dissolved with 0.2 ml pyridine adding 3 mg mannitol (as internal references) at 75 ◦ C for 30 min. After cooling to room temperature, hexamethyldisilazane (0.2 ml) and trimethylchlorosilane (0.1 ml) were added to the mixture. Then the trimethylsilylated derivatives were analyzed by GC (HP2010) using a RTX-WAX column and a flame ionization detector. The operation was performed using the following conditions—N2 : 0.8 ml/min; H2 : 1.5 ml/min; air: 200 ml/min; injection temperature: 200 ◦ C; detector temperature: 200 ◦ C. Sugar identification was done by comparison with reference sugars (rhamnose, fucose, arabinose, xylose, mannose, galactose, glucose, glucuronic acid, and galacturonic acid). 2.6. IR spectroscopy analysis For IR spectroscopy, the polysaccharide was mixed with KBr powder, ground and then pressed into a 1 mm pellets for Fourier transform infrared (FI-IR) measurement in the frequency range of 4000–500 cm−1 . FI-IR spectra of the polysaccharides were measured on a Nicolet Nexus870 spectrometer.
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buffer (150 mM, PH 7.4) for 30 min at 37 ◦ C, and hydroxyl radical was detected by monitoring absorbance at 520 nm. In the control, sample was substituted with distilled water, and sodium phosphate buffer replaced H2 O2 . The capability of scavenging hydroxyl radical was calculated using the following equation: scavenging effect (%) = [1 − A1 /A0 ] × 100, where A0 is the absorbance of the control and A1 is the absorbance of the sample. 2.8.3. Scavenging ability on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals The scavenging activity of the DPPH free radical was assayed according to the method of Kargozler et al. [23] with slight modification. In brief, 2 ml of sample solution at different concentrations (0.2–1.4 mg/ml) was added to 2 ml 0.2 mM ethanol solution of DPPH, the reaction mixture was shaken vigorously and incubated for 30 min in the dark at room temperature. The absorbance of the resulting solution was measured at 517 nm. The ability of scavenging the DPPH radicals was calculated using the following equation: scavenging effect (%) = [(A0 − A1 )/A0 ] × 100, where A0 is the absorbance of DPPH solution without the tested samples and A1 is the absorbance of the tested samples with DPPH solution. 2.8.4. Metal chelating assay The ferrous ion chelating effects of all samples were investigated according to the method reported by Liu et al. [24]. Briefly, the reaction mixture, containing different concentrations of samples (0.2–2.0 mg/ml), FeCl2 (0.1 ml, 2 mM), and ferroizine (0.4 ml, 5 mM), was shaken well and incubated for 10 min at room temperature. The absorbance of the mixture was measured at 562 nm against a blank. The ability of different concentrations of samples to chelate ferrous ion was calculated using the following equation: chelating ability (%) = [(A0 − A1 )/A0 ] × 100, where A0 is the absorbance of the control reaction and A1 is the absorbance of the sample. 2.9. Statistical analysis
2.7. De-sulfation Partial solvolytic de-sulfation was carried out as previously described [20]. The polysaccharides S1 and S2 in the pyridinium salt form were treated with 89:10:1 Me2 SO–MeOH–pyridine at 100 ◦ C for 8 h. The de-sulfated polysaccharides were recovered after dialysis and freeze-drying to afford DS-1 and DS-2.
All bioassay results were expressed as means ± standard deviation (SD). The experimental data were subjected to an analysis of variance (ANOVA) for a completely random design. For each polysaccharide, three samples were prepared for assays of every antioxidant attribute. 3. Results and discussion
2.8. Determination of antioxidant activity 3.1. Isolation and purification of the polysaccharide 2.8.1. Scavenging ability on superoxide radical The scavenging effect on superoxide radicals was assessed according to the method of Marklund and Marklund [21] with a minor modification. Briefly, 3 ml 0.05 mol/l Tris–HCl buffer (pH 8.2) and 1 ml of samples at different concentrations (0.05–0.21 mg/ml) were incubated at 25 ◦ C for 10 min, then 200 l of pyrogallol at the same temperature were added to the mixture, and the reaction was proceeded at 25 ◦ C for 4 min. Finally, the reaction system was terminated by the addition of 0.5 ml of HCl. The absorbance of the mixture was measured at 320 nm against the blank. The percentage inhibition of superoxide anion radicals scavenging was calculated using the following formula: scavenging effect (%) = [1 − A1 /A0 ] × 100, where A0 is the absorbance of control without the tested samples and A1 is the absorbance in the presence of the tested samples. 2.8.2. Scavenging ability on hydroxyl radical Hydroxyl radical-scavenging activity was measured by the method of Smirnoff and Cumbes [22] with a minor modification. The reaction mixture, containing different samples (0.4–1.6 mg/ml), was incubated with 2 mM EDTA–Fe (0.5 ml), 3% H2 O2 (1 ml), and 360 g/ml crocus in 4.5 ml sodium phosphate
The crude polysaccharide was extracted from U. pinnitafida and then purified by DEAE-Sephadex A-25 column, and two fractions were obtained: S1 and S2 from 1.1 and 1.6 M NaCl elution, respectively (Fig. 1A). Almost no polysaccharide was detected in 0–1 or 2.0 M NaCl elution. The two polysaccharide fractions were, respectively, pooled, dialyzed and lyophilized. The yields of the two fractions were 10.05% and 57.35% S1 and S2, respectively, appeared as only a single and symmetrically sharp peak on Superdex-200 (1.6 cm × 60 cm) with the AKTA Purifier system, indicating the two polysaccharide fractions (S1 and S2) were homogenous polysaccharides (Fig. 1B and C). 3.2. Chemical analysis The chemical composition of the polysaccharide was given in Tables 1 and 2. The results showed the main chemical components of all samples were rhamnose and sulfate, along with uronic acid and a small amount of protein. The sulfated polysaccharides (S1 and S2) had the higher sulfate content than the de-sulfated polysaccharides (DS-1 and DS-2). The rhamnose content in S1, S2,
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Table 2 Monosaccharide composition of S1, S2, DS-1 and DS-2 (mol%). Component S1 S2 DS-1 DS-2 a
Rha 51.44 40.30 50.37 39.94
Xyl 25.34 17.66 24.81 20.33
Gal 10.28 34.48 14.25 32.45
Ara
Glc
Fuc
a
a
a
nd nda nda nda
nd nda nda nda
nd nda nda nda
Man a
nd nda nda nda
GlcA
GalA
12.94 7.56 10.57 7.28
nda nda nda nda
Not detectable below the limit at 0.001.
DS-1 and DS-2 was 40.54%, 33.50%, 75.71% and 73.72%, respectively From gas chromatography, rhamnose was the main sugar unit, accounting for 51.44, 40.30, 50.37 and 39.94 mol% of the total sugar in S1, S2, DS-1 and DS-2. Xylose (24.81–25.34 mol%) was the second main sugar in the sample S1 and DS-1, while galactose (32.45–34.48 mol%) was the second main sugar in S2 and DS-2. In addition to rhamnose, xylose and galactose, glucuronic acid was also seen in the samples, which was consistent with the results of carbazole–sulfuric acid reaction. These results demonstrated that the polysaccharide (S1 and S2) isolated from U. pinnitafida had a different chemical composition to Phaeophyta from other species, was high rhamonse-containing sulfated polysaccharide. 3.3. IR spectroscopy FT-IR spectra of the four polysaccharides were similar. The signals around 3410–3420 cm−1 were from the stretch vibration of O–H existed in the hydrogen bond of the molecules. Signals at 2940 cm−1 were from the stretch vibration of –CH; IR spectrum of the sulfated polysaccharide demonstrated several bands corresponding to sulfate ester: the peaks at 847 and 1260 cm−1 derived from the bending vibration of C–O–S of the sulfate in axial position and stretching vibration of S–O of sulfate, respectively. The signals around 1260 cm−1 of DS-1 and DS-2 were weaker than S1 and S2, which means the content of sulfate group was lower than S1 and S2. In addition, signals at 1620–1635 cm−1 were attributed to the asymmetric stretch vibration of COO− of uronic acids. 1430 cm−1 was due to the symmetric stretch vibrations of COO− and the stretch vibration of C–O within –COOH. 3.4. Scavenging ability on superoxide radicals Superoxide radical can be generated by pyrogallol autooxidation and it can produce a colored compound. Resulting from a color change from purple to yellow, the absorbance at 320 nm increased when the superoxide anion was scavenged by an antioxidant, which can represent the content of superoxide radicals and indicate the antioxidant activity of the sample [25]. As shown in Fig. 2, the scavenging effects of all tested samples on superoxide radicals
Fig. 2. Scavenging effect of S1, S2, DS-1 and DS-2 on superoxide radicals. Results were representative of three separated experiments.
Fig. 3. Scavenging effect of S1, S2, DS-1 and DS-2 on hydroxyl radicals. Results were representative of three separated experiments.
were shown significantly in a concentration-dependent manner. A significant increase of the scavenging activity was observed at the concentration range (50–100 g/ml) of the polysaccharides. At a concentration of 210 g/ml, the scavenging activities were 82.65%, 80.02%, 49.93% and 48.01% for S1, S2, DS-1, and DS-2, respectively. But the scavenging effect of vitamin C was only 69.2% at a concentration of 200 g/ml. Compared to this result, the sulfated polysaccharide had stronger scavenging activity for superoxide radical than Vitamin C. Moreover, S1 and S2 had significantly higher scavenging activities on superoxide radicals than DS-1 and DS-2. This demonstrated that the sulfate content affected their antioxidant activity, which was in accordance with Zhang et al. [26] and Wang et al. [27] that higher sulfate content showed greater scavenging effect of superoxide radical. Among different reactive oxygen species (ROS), superoxide is generated first [28]. Although superoxide is a relatively weak oxidant, it may decompose to form stronger ROS, such as singlet oxygen and hydroxyl radical, which initiate peroxidation of lipids. Further, superoxide is also known to initiate indirectly the lipid peroxidation as a result of H2 O2 formation, creating precursors of hydroxyl radicals [29]. Therefore, superoxide scavenging is extremely important to antioxidant work. Our date on the activity of scavenging superoxide radicals suggested that it was likely to contribute towards the observed antioxidant effect. 3.5. Scavenging ability on hydroxyl radicals Hydroxyl radical is considered to be a highly potent oxidant which can react with most biomacromolecules functioning in living cells and induce severe damage to the adjacent biomolecules. Thus, removing hydroxyl radical is important for antioxidant defense in cell or food systems. As shown in Fig. 3, the two sulfated polysaccharides isolated from U. pinnitafida exhibited a strong scavenging activity on hydroxyl radicals. The scavenging ability of polysaccharides on hydroxyl radicals was in a concentration-dependent way. A significant increase of the scavenging effect was observed at the concentration range (0.4–1 mg/ml) of the samples. It was reported that scavenging effect on hydroxyl radicals of vitamin C was 50.0% and 60.0% at 1.63 mg/ml [30]. But the effects of S1 and S2 were 80.01% and 78.91% at 1.6 mg/ml, respectively. Moreover, all the sulfated polysaccharide fractions were found to have higher scavenging ability on hydroxyl radicals than de-sulfated samples. The present results proved that the sulfated polysaccharides isolated from U. pinnitafida were good scavengers for hydroxyl radicals and the sulfate group played an important role in the scavenging of hydroxyl radicals. Previous studies had reported that there are two type of antioxidation mechanism: one suppresses the generation of the hydroxyl
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Fig. 4. Scavenging effect of S1, S2, DS-1 and DS-2 on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. Results were representative of three separated experiments.
radical and the other scavenges the hydroxyl radicals generated. In the former, the antioxidant activity may ligate to the metal ions which H2 O2 to give the metal complexes. The metal complexes thus formed cannot further react with H2 O2 to give a hydroxyl radical [31,32]. In another assay system in this study, it was demonstrated that the iron chelating ability and the trend of the chelating ability were nearly similar to the order of the scavenging ability to hydroxyl radical. The antioxidant activities of the tested samples were not a function of a single factor but a combination of several factors. 3.6. Free radical-scavenging activity on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical The DPPH free radical is a stable free radical, which has been widely accepted as a tool for estimating the free radical-scavenging activities of antioxidants [33]. When DPPH free radical encounters a proton-donating substance, for example, an antioxidant, the radical would be scavenged and the absorbance at 517 nm is reduced. Based on this principle, the antioxidant activity of a substance can be expressed as its ability in scavenging the DPPH free radical [34]. As shown in Fig. 4, the scavenging abilities of all samples were in a concentration-dependent fashion. At a concentration of 0.6 mg/ml, the four polysaccharides showed scavenging abilities of 4.9–20.21% on DPPH radicals. At 1.4 mg/ml, the scavenging effect increased to 30.87%, 35.01%, 56.12% and 60.88% for S1, S2, DS-1 and DS-2, respectively. Apparently, S1 and S2 had higher scavenging abilities than DS-1 and DS-2. However, the scavenging effects of the four polysaccharides on DPPH radicals were all relatively lower than that of ascorbic acid at the same concentration. 3.7. Chelating effect on ferrous ions Fig. 5 showed ferrous ion chelating effects of S1, S2 and DS-2 were concentration related and that of DS-1 was not concentration dependent. At a concentration of 0.2 mg/ml, the chelating effect of DS-1 was stronger than that of high content polysaccharide, but it was not significant. At high concentration, the effects of S1 and S2 were more pronounced than that of de-sulfated samples, and the effect of DS-1 was the lowest. Metal chelating activity is claimed as one of antioxidant mechanisms, since it reduces the concentration of the catalyzing transition metal in lipid peroxidation. Among the transition metals, iron is known as the most important lipid oxidation prooxidant due to its high reactivity. The ferrous state of iron accelerates lipid oxidation by breaking down hydrogen and lipid peroxides to reactive free radicals via the Fenton reaction
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Fig. 5. Chelating ability of S1, S2, DS-1 and DS-2 on ferrous ions. Results were representative of three separated experiments.
(Fe2+ + H2 O2 → Fe3+ + OH− + OH• ). Fe3+ ion also produces radicals from peroxides although the rate is less than tenth that of Fe2+ ion. Thus Fe2+ ion is the most powerful prooxidant among the various species of metal ions [35]. The date of Fe2+ chelating effect demonstrated that the sulfated polysaccharides S1 and S2 exhibited moderate chelating activity. 4. Conclusion According to the results stated above, it could be concluded the crude polysaccharide extracted from U. pinnitafia predominantly contained two polysaccharide fractions (S1 and S2) after purified by DEAE-Sephadex A-25 column chromatography. The study also showed two polysaccharide fractions were high rhamnosecontaining sulfated polysaccharides. In addition, S1 and S2 were antioxidative and exhibited stronger antioxidant activities compared to that of de-sulfated polysaccharides in vitro. A positive correlation has been revealed between sulfate content and antioxidant activity. Overall, the present experiments on bioactivity of the sulfated polysaccharide from U. pinnitafia showed it was useful as functional food as well as potential therapeutic agent. Acknowledgement This work was supported by the National Natural Science Foundation of China (30800194). References [1] G. Blander, R.M. Oliveira, C.M. Conboy, M. Haigis, L. Guarente, J. Biol. Chem. 278 (2003) 38966–38969. [2] F. Liu, V.E.C. Ooi, S.T. Chang, Life Sci. 60 (1997) 763–771. [3] J.L. Mau, H.C. Lin, C.C. Chen, J. Agric. Food Chem. 50 (2002) 6072–6077. [4] H.C. Grice, Food Chem. Toxicol. 26 (1988) 717–723. [5] S. Nandita, P.S. Rajini, Food Chem. 85 (2004) 611–616. [6] Q.B. Zhang, N. Li, X.G. Liu, Z.Q. Zhao, Z.H. Xu, Carbohydr. Res. 339 (2004) 105–111. [7] T. Nishino, T. Nagumo, Carbohydr. Res. 229 (1992) 355–362. [8] S. Koyanagi, N. Tanigawa, H. Nakagawa, S. Soeda, H. Shimeno, Biochem. Pharamacol. 65 (2003) 173–179. [9] N.M.A. Ponce, C.A. Pujol, E.B. Damonte, M.L. Flores, C.A. Stoerz, Carbohydr. Res. 338 (2003) 153–165. [10] H. Qi, Q. Zhang, T. Zhao, R. Hu, K. Zhang, Z. Li, Bioorg. Med. Chem. Lett. 16 (2006) 2441–2445. [11] H.J. Zhang, W.J. Mao, F. Fang, H.Y. Li, H.H. Sun, Y. Chen, X.H. Qi, Carbohydr. Polym. 71 (2008) 428–434. [12] J.B. Lee, K. Hayashi, M. Maeda, T. Hayashi, Planta Med. 65 (1999) 439–441. [13] S.R. Sem, A.K. Das, A.K. Siddhanta, K.H. Mody, B.K. Ramavat, V.D. Chauhan, J.R. Vedasiromoni, D.K. Ganguly, Int. J. Biol. Macromol. 16 (1994) 279–280. [14] M.R.S. Melo, J.P.A. Feitosa, A.L.P. Freitas, R.C.M. de Paula, Carbohydr. Polym. 49 (2002) 491–498. [15] D.J. Schaeffer, V.S. Krylov, Ecotoxicol. Environ. Safety 45 (2000) 208–227.
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