- Email: [email protected]
Free-radical-scavenging activity and antioxidant effect of ascophyllan from marine brown algae Padina tetrastromatica
G Model BIONUT-177; No. of Pages 5
ARTICLE IN PRESS Biomedicine & Preventive Nutrition xxx (2013) xxx–xxx
Available online at www.sciencedirect.com
Original article
Free-radical-scavenging activity and antioxidant effect of ascophyllan from marine brown algae Padina tetrastromatica S. Mohsin , R. Mahadevan , G. Muraleedhara Kurup ∗ Department of Biochemistry, University of Kerala, Kariavattom Campus, Thiruvananthapuram, 695581, Kerala, India
a r t i c l e
i n f o
Article history: Received 19 July 2013 Accepted 9 August 2013 Keywords: Sulfated polysaccharide Antioxidant activity Radical-scavenging effect Reducing power Padina tetrastromatica
a b s t r a c t Brown seaweeds are rich in sulfated polysaccharides that could potentially be exploited as functional ingredients for human health. Over the years, sulfated polysaccharides with potential pharmacological, nutraceutical and functional food properties have been isolated from brown seaweeds. This study was designed to characterise and evaluate the antioxidant activity of a sulfated polysaccharide ascophyllan isolated from marine brown algae Padina tetrastromatica. Four polysaccharide fractions were purified from crude ascophyllan. Antioxidant activities of the polysaccharide fractions were evaluated by various in vitro assay systems, including 2,2-diphenyl-1-picrylhydrazyl (DPPH), superoxide anion, hydroxyl radical-scavenging activity, lipid peroxidation and reducing power assay. The results showed that the ascophyllan fraction AF3 showed stronger free-radical-scavenging abilities and had good antioxidant effect. Available data obtained by in vitro models suggest that there is a correlation between the sulfate content and antioxidant activity. © 2013 Elsevier Masson SAS. 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]. Therefore, antioxidants are important for protection against oxidative stress. Lipid oxidation by reactive oxygen species (ROS), such as super oxide anion, hydroxyl radicals and hydrogen peroxide also cause a decrease in nutritional value of lipids, in their safety and appearance. Recently, there is a considerable interest in the food industry and in the preventive medicine for the development of antioxidants from natural sources, such as marine flora and fauna, including bacteria, fungi and higher plants. Among them, marine algae represent one of the richest sources of bioactive compounds and algae-derived products are increasingly used in medical and biochemical research [4]. One particularly interesting feature of marine algae is their richness in sulfated polysaccharides [5]. In marine algae, they occur as sulfated fucans and sulphated galactans. Sulfated polysaccharides from marine algae contain diverse biological activities with potential medicinal value, such as anticoagulant,
∗ Corresponding author. Tel.: +91 47 12308078; fax: +91 47 12308078. E-mail address: [email protected] (G. Muraleedhara Kurup).
antitumor, antiviral and antioxidant [6–12]. Their activity depends on several structural parameters, such as the degree of sulfation, the molecular weight and type of sugar [13]. Padina tetrastromatica is a marine brown algae found in abundance in the coastal areas of India. The uses and potential values of ascophyllan from P. tetrastromatica have not been well studied so far and no reports of antioxidant potential. So, in the present study, the antioxidative effect of the polysaccharide was examined in detail and the results are discussed. 2. Materials and methods 2.1. Chemicals and solvents All biochemicals used in this study were purchased from Sigma chemical company, St. Louis, MO, USA and all the other chemicals used were of highest grade available. 2.2. Sea weed material Samples of P. tetrastromatica were collected from the Vizhinjam ◦ ◦ coast of Kerala, located at 8 21 N and 77 0 E on the west coast of India. The algal material was identified by the Botanist (Dr. M.V.N Panikkar, Professor, Department of Botany, S.N. College, Kollam, Kerala and Dr. G. Valsala Devi, Professor, Department of Botany, University of Kerala) and a voucher herbarium specimen (No. KUBH 5804) was deposited in the herbarium of Department of Botany, University of Kerala, India. The seaweeds were washed thoroughly
2210-5239/$ – see front matter © 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.bionut.2013.08.006
Please cite this article in press as: Mohsin S, et al. Free-radical-scavenging activity and antioxidant effect of ascophyllan from marine brown algae Padina tetrastromatica. Biomed Prev Nutr (2013), http://dx.doi.org/10.1016/j.bionut.2013.08.006
G Model BIONUT-177; No. of Pages 5
ARTICLE IN PRESS S. Mohsin et al. / Biomedicine & Preventive Nutrition xxx (2013) xxx–xxx
2
with tap water, dried by forced air circulation and pulverized in a warring blender. Depigmentation of algal powder was done using sequential extraction with petroleum ether and acetone as solvent in a soxhlet apparatus. The residue material was air-dried to yield algal powder. 2.3. Extraction, purification and characterisation of ascophyllan fractions Ascophyllan was extracted, purified and characterised by the method of Mohsin et al. [14]. 2.4. Determination of antioxidant activity 2.4.1. 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 Karagozler et al. [15] with a slight modification. In brief, 2 mL of ascophyllan fractions of different concentrations (10–50 g/mL) were 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. Values of the concentration of samples required to scavenge 50% of free-radicals or to prevent lipid peroxidation by 50% (IC50 ) were calculated from the regression equations prepared from the concentration of samples and percentage inhibition of each system. 2.4.2. Scavenging ability on superoxide radical Superoxide radical-scavenging activity was determined by the method of Robak and Gryglewski [16]. Superoxide radical was generated in the PMS-NADH system containing 3 mL Tris–HCl buffer (16 mM, pH 8.0), 338 M NADH, 72 M NBT, 30 M PMS and varying concentrations of ascophyllan fractions (10–50 g/mL). The mixture prepared earlier was incubated at room temperature for 5 min and the absorbance was read at 560 nm against the blank. In the control, the sample was substituted with Tris–HCl buffer. 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. 2.4.3. Hydroxyl radical-scavenging assay Hydroxyl radical-scavenging activity was measured using a modified Smironoff and Cumbes’ method [17]. The reaction mixture, containing ascophyllan fractions of different concentrations (10–50 g/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 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, the sample was substituted with distilled water, and sodium phosphate buffer was replaced with H2 O2 . 2.4.4. Liver microsomal lipid peroxidation The effects of polysaccharides on lipid peroxidation were determined according to Liu et al. [18]. Liver microsomes were prepared from Wistar rats. Liver was homogenized in ice-cold 0.25 M sucrose and then centrifuged at 12,000 × g for 20 min at 4 ◦ C. The supernatant obtained was centrifuged at 105,000 × g for 60 min at 4 ◦ C. The microsomes were washed with ice-cold 0.15 M KCl, and then
stored at −20 ◦ C. The lipid peroxidation assay was performed in Fe2+ /vitamin C system. The microsomes (300 g/mL) were incubated at 37 ◦ C for 60 min with varying concentrations of ascophyllan fractions (10–50 g/mL), 10 mM FeS04 ·7H2 O and 0.1 mM ascorbic acid in 1.0 mL potassium phosphate buffer (0.2 M, pH 7.4). The reaction was stopped by the addition of 20% trichloroacetic acid (1.0 mL) and 0.67% 2-thiobarbituric acid (TBA) (1.5 mL) in succession, and the solution was then heated at 100 ◦ C for 15 min [19]. The condensation reaction occurring between the MDA and TBA produces a pink compound, which has a strong absorption at 532 nm [20]. The percentage of antioxidant activity of the samples was evaluated according to the following formula [21]. Inhibition (rate %) = (A0 − A)/(A0 − Ae ) × 100, where A0 is the absorbance of the free-radical generation system, A is the absorbance of the test sample and Ae is the absorbance of the essential control. 2.4.5. Reducing power assay The reducing power was determined as described by Yen and Chen [22]. Briefly, 0.13 mL of ascophyllan fractions of different concentrations (10–50 g/mL) in phosphate buffer (0.2 M, pH 6.6) were mixed with 0.125 mL of potassium ferricyanide (1%, w/v) and incubated at 50◦ C for 20 min. Afterwards, 0.125 mL of TCA (10%, w/v) were added to the mixture to terminate the reaction. Then, the solution was mixed with 1.5 mL ferric chloride (0.1%, w/v) and the absorbance was measured at 700 nm. 2.5. Statistical analysis The results were analyzed using a statistical program SPSS/PC+, version 11.0 (SPSS Inc., Chicago, IL, USA). Statistical evaluation was done using the one-way ANOVA and significant difference was determined using Duncans test at the level of P < 0.05. 3. Results 3.1. Purification of ascophyllan The total yield of crude ascophyllan (CA) fraction from P. tetrastromatica was 17%. The isolated CA was fractionated by DEAE Sepharose column chromatography using phosphate buffer containing NaCl (0.0–4.0 M) gradient as the eluent. Four fractions namely, ascophyllan fraction1 (AF1) eluted with 0.17 to 0.86 M gradient, ascophyllan fraction 2 (AF2) eluted with 1.25 to 2.05 M gradient, ascophyllan fraction 3 (AF3) eluted with 2.48 to 2.96 M gradient and ascophyllan fraction 4 (AF4) eluted with 3.07 to 3.45 M gradient, respectively. Chemical composition showed that the AF3 fraction contained 42.13% neutral sugars, 15.3% sulphate, 23.5% uronic acid and 0.9% proteins [14]. 3.2. Free-radical-scavenging activity of ascophyllan fractions on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical Fig. 1 shows the DPPH radical-scavenging activity of the ascophyllan fractions from P. tetrastromatica. The scavenging activity of AF1, AF2 and AF4 were 31%, 56% and 16% at 50 g/mL concentration. The AF3 fraction showed an IC50 of 24.7 g/mL while the AF2 fraction showed 39.2 g/mL. The IC50 of AF1 and AF4 was not significant when compared to AF3 and AF2 fractions. However, the IC50 of quercetin, a known free-radical scavenger was only 19.75 ± 1.30 g/mL. 3.3. Superoxide anion scavenging activity of ascophyllan fractions The IC50 values of AF2, AF3 and quercetin were 47.2, 38.9 and 32.3 g/mL; however, the IC50 values of AF1 and AF4 was not much
Please cite this article in press as: Mohsin S, et al. Free-radical-scavenging activity and antioxidant effect of ascophyllan from marine brown algae Padina tetrastromatica. Biomed Prev Nutr (2013), http://dx.doi.org/10.1016/j.bionut.2013.08.006
G Model BIONUT-177; No. of Pages 5
ARTICLE IN PRESS S. Mohsin et al. / Biomedicine & Preventive Nutrition xxx (2013) xxx–xxx
Fig. 1. Scavenging effect of ascophyllan fractions on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. Values are expressed as average of three samples ± SEM.
3
Fig. 3. Scavenging effect of ascophyllan fractions on hydroxyl radicals. Values are expressed as average of three samples ± SEM.
significant. Compared with this result, AF3 showed a stronger scavenging activity for superoxide radicals than other fractions. AF2 and AF3 fraction showed scavenging effect of 53.4% and 75.6% at 50 g/mL concentration. The scavenging effect of the AF1 and AF4 was less than 50%. The results are shown in Fig. 2. 3.4. Hydroxyl radical-scavenging activity of ascophyllan fractions The hydroxyl radical-scavenging effect of ascophyllan fractions and quercetin is shown in Fig. 3. Among the four samples and quercetin, AF3 had stronger scavenging effect against hydroxyl radical. At 50 g/mL, AF3 fraction showed 88.4% scavenging effect while quercetin showed 85.7%. The IC50 values of AF3 and quercetin were 31.2 g/mL and 27.3 g/mL while the IC50 values of AF1, AF2 and AF4 were not much significant. 3.5. Inhibitory effect of ascophyllan fractions on lipid peroxidation
Fig. 4. Inhibition of lipid peroxidation of rat liver microsome by ascophyllan fractions. Values are expressed as average of three samples ± SEM.
3.6. Reducing power of ascophyllan fractions Inhibition of ascophyllan fractions on lipid peroxidation of rat liver microsomes is shown in Fig. 4. The free-radical formation in this system was inhibited by all of the polysaccharide samples with IC50 values of 44.2, 28.2 and 35.2 g/mL for AF2, AF3 and quercetin respectively. At 50 g/mL, AF3 showed 90% inhibition which was similar to quercetin at the same dose.
Fig. 5 depicts the reducing power of tested samples. Higher absorbance value means stronger reducing power of samples. Though the reducing power of AF1 and AF4 was low in the tested concentration, but AF2 and AF3 fractions were concentrationdependent. The reducing power of AF1 and AF4 at 50 g/mL were
Fig. 2. Scavenging effect of ascophyllan fractions on superoxide radicals. Values are expressed as average of three samples ± SEM.
Fig. 5. Reducing power of ascophyllan fractions. Values are expressed as average of three samples ± SEM.
Please cite this article in press as: Mohsin S, et al. Free-radical-scavenging activity and antioxidant effect of ascophyllan from marine brown algae Padina tetrastromatica. Biomed Prev Nutr (2013), http://dx.doi.org/10.1016/j.bionut.2013.08.006
G Model BIONUT-177; No. of Pages 5
ARTICLE IN PRESS S. Mohsin et al. / Biomedicine & Preventive Nutrition xxx (2013) xxx–xxx
4
0.027 and 0.016 respectively, which were much weaker than those of AF2 and AF3. Moreover, the reducing power of quercetin at 50 g/mL was 0.135.
4. Discussion Marine algae are an abundant source of new bioactive natural compounds with a wide range of important biological activities, such as antioxidant, anticoagulant and antithrombotic activities [23]. P. tetrastromatica has been traditionally used in India as a functional food for centuries. In this context, we isolated and characterized a water soluble sulphated polysaccharide from P. tetrastromatica and examined its antioxidant activity. Extraction of algae yielded Crude Ascophyllan (CA), which was approximately 17%. Fractionation of CA by anion exchange column chromatography gave four fractions. 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 [24]. When DPPH free-radical encounters 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 [25] The DPPH radical-scavenging activity of ascophyllan fractions from P. tetrastromatica has been attributed to the ability of the polysaccharide fractions in pairing with the odd electron of DPPH radical [26]. Among them, AF3 fraction showed maximum scavenging activity. Higher radical-scavenging activity was observed in fractions when compared to crude extract [27]. Superoxide radical can be generated by autooxidation and it can produce a coloured compound. Resulting from a colour 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 [28]. The scavenging ability of the ascophyllan fractions decreased in the order of AF3 > AF2 > AF1 > AF4 corresponding to the sulfate content of the samples in the order of 15.3, 6.59, 2.56 and 0.15%, respectively[14]. This clearly demonstrates that the sulfate content affected the antioxidant activity and these results are in agreement with the observations of Qi et al. [29] and Tsiapali et al. [30] who found that sulfated glucan exhibited greater antioxidant ability. Among different reactive oxygen species (ROS), superoxide is generated first [31]. 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 [32]. Our data on the activity of superoxide radical-scavenging suggested that it was likely to contribute towards the observed antioxidant effect. Among the reactive oxygen species, the hydroxyl radical is the most reactive and induces severe damage to adjacent biomolecules. The result suggests that AF3 had the strongest scavenging ability for hydroxyl radicals. Previous studies of the antioxidant activity of various natural plant derived biomolecules have reported that the scavenging activity for hydroxyl radicals is not due to direct scavenging but due to inhibition of hydroxyl radical generation by chelating ions, such as Fe2+ and Cu+ [33]. Lipid peroxidation is a key process in many pathological events and is one of the reactions induced by oxidative stress. The unsaturated fatty acids in cell membrane on oxidation lead to the formation and proliferation of lipid peroxides. The oxygen uptake, structural rearrangements of unsaturated fatty acids and ultimate damage of membrane lipids lead to production of malondialdehyde, which is known to be carcinogenic and mutagenic[34]. The inhibition of lipid peroxidation by AF3 might probably be due to its
free-radical-scavenging potential and thereby proves to be a good antioxidant and cytoprotective agent. It has been reported that reducing power serves as a significant reflection of the antioxidant activity. The presence of reductants in the antioxidant samples cause the reduction of the Fe3+ /ferricyanide complex to the ferrous form. The reducing properties were generally associated with the presence of reductones, which have been shown to exert antioxidant activity by breaking the free-radical chain by donating a hydrogen atom. Most non-enzymatic antioxidative activity, such as scavenging of freeradicals or inhibition of peroxidation, is mediated by redox reaction [35]. Our data shows that reducing power of ascophyllan fractions play a significant role as an antioxidant. 5. Conclusion The results clearly demonstrate that sulfated polysaccharide extracted from P. tetrastromatica was a good antioxidant. AF3 fraction was chosen for further investigation of its antioxidant activities and mechanisms in vivo in our future work. Overall, the present experiments showed that ascophyllan from marine brown algae was a potential therapeutic agent. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgement Financial assistance from UGC in the form of Research Fellowship in Science to Meritorious Students (RFSMS) is gratefully acknowledged. References [1] Blander G, Oliveira RM, Conboy CM, Haigis M, Guarente L. Superoxide dismutase 1 knock-down induces senescence in human fibroblasts. J Biol Chem 2003;278:38966–9. [2] Liu F, Ooi VEC, Chang ST. Free radical scavenging activity of mushroom polysaccharide extracts. Life Sci 1997;60:763–71. [3] Mau J-L, Lin H-C, Chen C-C. Antioxidant properties of several medicinal mushrooms. J Agric Food Chem 2002;50(21):6072–7. [4] Mayer AMS, Lehmann VKB. Marine pharmacology in 1998: marine compounds with antibacterial, anticoagulant, antifungal, anti-inflammatory, anthelmintic, antiplatelet, antiprotozoal and antiviral activities; with actions on the cardiovascular, endocrine, immune and nervous systems: And other miscellaneous mechanisms of action. Pharmacologist 2000;42:62–9. [5] Renn DW. Biotechnology and the red seaweed polysaccharide industry: status, needs and prospects. Trends Biotechnol 1997;15:9–14. [6] Nishino T, Nagumo T. Anticoagulant and antithrombin activities of oversulfated fucans. Carbohydr Res 1992;229:355–62. [7] Koyanagi S, Tanigawa N, Nakagawa H, Soeda S, Shimeno H. Oversulfation of fucoidan enhances its anti-angiogenic and antitumor activities. Biochem Pharmacol 2003;65(2):173–9. [8] Ponce NMA, Pujol CA, Damonte EB, Flores ML, Stortz CA. Fucoidans from the brown seaweed Adenocystis utricularis: extraction methods, antiviral activity and structural studies. Carbohydr Res 2003;338:153–65. [9] Qi H, Zhang Q, Zhao T, Hu R, Zhang K, Li Z. In vitro antioxidant activity of acetylated and benzoylated derivatives of polysaccharide extracted from Ulva pertusa (Chlorophyta). Bioorg Med Chem Lett 2006;16(9):2441–5. [10] Zhang HJ, Mao WJ, Fang F, Li HY, Sun HH, Chen Y, et al. Chemical characteristics and anticoagulant activities of a sulfated polysaccharide and its fragments from Monostroma latissimum. Carbohydr Polym 2008;71:428–34. [11] Lee JB, Hayashi K, Hayashi T, Sankawa U, Maeda M. Antiviral activities against HSV-1, HCMV, and HIV-1 of rhamnan sulfate from Monostroma latissimum. Planta Med 1999;65(5):439–41. [12] Sem SR, Das AK, Siddhanta AK, Mody KH, Ramavat BK, Chauhan VD, et al. A new sulphated polysaccharide with potent anti-coagulant activity from the red seaweed Grateloupia indic. Int J Biol Macromol 1994;16:279–80. [13] Melo MRS, Feitosa JPA, Freitas ALP, De Paula RCM. Isolation and characterisation of sulphated soluble polysaccharide from the red seaweed Gracilaria cornea. Carbohydr Polym 2002;49:491–8. [14] Mohsin S, Muraleedhara Kurup G, Mahadevan R. Effect of ascophyllan from brown algae Padina tetrastromatica on inflammation and
Please cite this article in press as: Mohsin S, et al. Free-radical-scavenging activity and antioxidant effect of ascophyllan from marine brown algae Padina tetrastromatica. Biomed Prev Nutr (2013), http://dx.doi.org/10.1016/j.bionut.2013.08.006
G Model BIONUT-177; No. of Pages 5
ARTICLE IN PRESS S. Mohsin et al. / Biomedicine & Preventive Nutrition xxx (2013) xxx–xxx
[15] [16] [17] [18] [19] [20]
[21]
[22] [23]
[24] [25]
oxidative stress in carrageenan-induced rats. Inflammation 2013, http://dx.doi.org/10.1007/s10753-013-9665-4. Karagozler AA, Erday B, Emek YC, Uggun DA. Antioxidant activity and proline content of leaf extracts from Dorystoechas hastata. Food Chem 2008;111:400–7. Robak J, Gryglewski IR. Flavonoides are scavengers of superoxide anions. Biochem Pharmacol 1988;37:837–41. Smironoff CQ. Hydroxyl radical-scavenging activity of compatible solutes. Phytochemistry 1989;28:1057–60. Liu F, Ooi VCE, Chang ST. Free radical scavenging activities of mushroom polysaccharide extracts. Life Sci 1997;60:763–71. Bueg JA, Aust SD. Microsomal lipid peroxidation. In: Feischer S, Packer L, editors. Methods in Enzymology, 52. New York: Academic Press; 1978. p. 302–10. Wei Z, Bai O, Steven Richardson J, Mousseau DD, Li X. Olanzapine protects PC12 cells from oxidative stress induced by hydrogen peroxide. J Neurosci Res 2003;73:364–8. Zhang Q, Yu P, Li Z, Zhang H, Xu Z, Li P. Antioxidant activities of sulfated polysaccharide fractions from Porphyra haitanesis. J Appl Phycol 2003;15: 305–10. Yen GC, Chen HY. Antioxidant activity of various tea extracts in relation to their antimutagenicity. J Agric Food Chem 1995;43:27–32. Shanmugam M, Mody KH. Heparinoid-active sulphated polysaccharides from marine algae as potential blood anticoagulant agents. Curr Sci 2000;79:1672–83. Huang D, Ou B, Prior RL. The chemistry behind antioxidant capacity assays. J Agric Food Chem 2005;53:1841–56. Li XL, Zhou AG, Li XM. Inhibition of Lycium barbarum polysaccharides and Ganoderma lucidum polysaccharides against oxidative injury induced by irradiation in rat liver mitochondria. Carbohydr Polym 2007;69:172–8.
5
[26] Park PJ, Shahidi F, Jeon YJ. Antioxidant activities of enzymatic extracts from edible seaweed Sargassum horneri using ESR spectroscopy. J Food Lipids 2004;11:15–27. [27] Duan XJ, Zhang W, Li XM, Wang BG. Evaluation of antioxidant property of extract and fractions obtained from a red alga, Polysiphonia urceolata. Food Chem 2006;95:37–43. [28] Chen Y, Xie MY, Nie SP, Li C, Wang YX. Purification, composition analysis and antioxidant activity of a polysaccharide from the fruiting bodies of Ganoderma atrum. Food Chem 2008;107:231–41. [29] Qi H, Zhang Q, Zhao T, Chen R, Zhang H, Niu X, et al. Antioxidant activity of different sulfate content derivatives of polysaccharide extracted from Ulva pertusa (Chlorophyta) in vitro. Int J Biol Macromol 2005;37:195–9. [30] Tsiapali E, Whaley S, Kalbfleisch J, Ensley HE, Browder IW, Williams DL. Glucans exhibit weak antioxidant activity, but stimulate macrophage free radical activity. Free Radic Biol Med 2001;30:393–402. [31] Liu F, Ng TB. Antioxidative and free radical scavenging activities of selected medicinal herbs. Life Sci 2000;66:725–35. [32] Wade CR, Jackson PG, Highton J, van Rij AM. Lipid peroxidation and malondialdehyde in the synovial fluid and plasma of patients with rheumatoid arthritis. Clin Chim Acta 1987;164:245–50. [33] Halliwell B, Gutteridge JMC, Aruoma OI. The deoxyribose method: a simple “test-tube” assay for determination of rate constants for reactions of hydroxyl radicals. Anal Biochem 1987;165:215–9. [34] Miyake T, Shibamoto T. Inhibition of malonaldehyde and acetaldehyde formation from blood plasma oxidation by naturally occurring antioxidants. J Agric Food Chem 1998;46:3694–7. [35] Zhu QY, Hackman RM, Ensunsa JL, Holt RR, Keen CL. Antioxidative activities of oolong tea. J Agric Food Chem 2002;50:6929–34.
Please cite this article in press as: Mohsin S, et al. Free-radical-scavenging activity and antioxidant effect of ascophyllan from marine brown algae Padina tetrastromatica. Biomed Prev Nutr (2013), http://dx.doi.org/10.1016/j.bionut.2013.08.006
ARTICLE IN PRESS Biomedicine & Preventive Nutrition xxx (2013) xxx–xxx
Available online at www.sciencedirect.com
Original article
Free-radical-scavenging activity and antioxidant effect of ascophyllan from marine brown algae Padina tetrastromatica S. Mohsin , R. Mahadevan , G. Muraleedhara Kurup ∗ Department of Biochemistry, University of Kerala, Kariavattom Campus, Thiruvananthapuram, 695581, Kerala, India
a r t i c l e
i n f o
Article history: Received 19 July 2013 Accepted 9 August 2013 Keywords: Sulfated polysaccharide Antioxidant activity Radical-scavenging effect Reducing power Padina tetrastromatica
a b s t r a c t Brown seaweeds are rich in sulfated polysaccharides that could potentially be exploited as functional ingredients for human health. Over the years, sulfated polysaccharides with potential pharmacological, nutraceutical and functional food properties have been isolated from brown seaweeds. This study was designed to characterise and evaluate the antioxidant activity of a sulfated polysaccharide ascophyllan isolated from marine brown algae Padina tetrastromatica. Four polysaccharide fractions were purified from crude ascophyllan. Antioxidant activities of the polysaccharide fractions were evaluated by various in vitro assay systems, including 2,2-diphenyl-1-picrylhydrazyl (DPPH), superoxide anion, hydroxyl radical-scavenging activity, lipid peroxidation and reducing power assay. The results showed that the ascophyllan fraction AF3 showed stronger free-radical-scavenging abilities and had good antioxidant effect. Available data obtained by in vitro models suggest that there is a correlation between the sulfate content and antioxidant activity. © 2013 Elsevier Masson SAS. 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]. Therefore, antioxidants are important for protection against oxidative stress. Lipid oxidation by reactive oxygen species (ROS), such as super oxide anion, hydroxyl radicals and hydrogen peroxide also cause a decrease in nutritional value of lipids, in their safety and appearance. Recently, there is a considerable interest in the food industry and in the preventive medicine for the development of antioxidants from natural sources, such as marine flora and fauna, including bacteria, fungi and higher plants. Among them, marine algae represent one of the richest sources of bioactive compounds and algae-derived products are increasingly used in medical and biochemical research [4]. One particularly interesting feature of marine algae is their richness in sulfated polysaccharides [5]. In marine algae, they occur as sulfated fucans and sulphated galactans. Sulfated polysaccharides from marine algae contain diverse biological activities with potential medicinal value, such as anticoagulant,
∗ Corresponding author. Tel.: +91 47 12308078; fax: +91 47 12308078. E-mail address: [email protected] (G. Muraleedhara Kurup).
antitumor, antiviral and antioxidant [6–12]. Their activity depends on several structural parameters, such as the degree of sulfation, the molecular weight and type of sugar [13]. Padina tetrastromatica is a marine brown algae found in abundance in the coastal areas of India. The uses and potential values of ascophyllan from P. tetrastromatica have not been well studied so far and no reports of antioxidant potential. So, in the present study, the antioxidative effect of the polysaccharide was examined in detail and the results are discussed. 2. Materials and methods 2.1. Chemicals and solvents All biochemicals used in this study were purchased from Sigma chemical company, St. Louis, MO, USA and all the other chemicals used were of highest grade available. 2.2. Sea weed material Samples of P. tetrastromatica were collected from the Vizhinjam ◦ ◦ coast of Kerala, located at 8 21 N and 77 0 E on the west coast of India. The algal material was identified by the Botanist (Dr. M.V.N Panikkar, Professor, Department of Botany, S.N. College, Kollam, Kerala and Dr. G. Valsala Devi, Professor, Department of Botany, University of Kerala) and a voucher herbarium specimen (No. KUBH 5804) was deposited in the herbarium of Department of Botany, University of Kerala, India. The seaweeds were washed thoroughly
2210-5239/$ – see front matter © 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.bionut.2013.08.006
Please cite this article in press as: Mohsin S, et al. Free-radical-scavenging activity and antioxidant effect of ascophyllan from marine brown algae Padina tetrastromatica. Biomed Prev Nutr (2013), http://dx.doi.org/10.1016/j.bionut.2013.08.006
G Model BIONUT-177; No. of Pages 5
ARTICLE IN PRESS S. Mohsin et al. / Biomedicine & Preventive Nutrition xxx (2013) xxx–xxx
2
with tap water, dried by forced air circulation and pulverized in a warring blender. Depigmentation of algal powder was done using sequential extraction with petroleum ether and acetone as solvent in a soxhlet apparatus. The residue material was air-dried to yield algal powder. 2.3. Extraction, purification and characterisation of ascophyllan fractions Ascophyllan was extracted, purified and characterised by the method of Mohsin et al. [14]. 2.4. Determination of antioxidant activity 2.4.1. 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 Karagozler et al. [15] with a slight modification. In brief, 2 mL of ascophyllan fractions of different concentrations (10–50 g/mL) were 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. Values of the concentration of samples required to scavenge 50% of free-radicals or to prevent lipid peroxidation by 50% (IC50 ) were calculated from the regression equations prepared from the concentration of samples and percentage inhibition of each system. 2.4.2. Scavenging ability on superoxide radical Superoxide radical-scavenging activity was determined by the method of Robak and Gryglewski [16]. Superoxide radical was generated in the PMS-NADH system containing 3 mL Tris–HCl buffer (16 mM, pH 8.0), 338 M NADH, 72 M NBT, 30 M PMS and varying concentrations of ascophyllan fractions (10–50 g/mL). The mixture prepared earlier was incubated at room temperature for 5 min and the absorbance was read at 560 nm against the blank. In the control, the sample was substituted with Tris–HCl buffer. 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. 2.4.3. Hydroxyl radical-scavenging assay Hydroxyl radical-scavenging activity was measured using a modified Smironoff and Cumbes’ method [17]. The reaction mixture, containing ascophyllan fractions of different concentrations (10–50 g/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 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, the sample was substituted with distilled water, and sodium phosphate buffer was replaced with H2 O2 . 2.4.4. Liver microsomal lipid peroxidation The effects of polysaccharides on lipid peroxidation were determined according to Liu et al. [18]. Liver microsomes were prepared from Wistar rats. Liver was homogenized in ice-cold 0.25 M sucrose and then centrifuged at 12,000 × g for 20 min at 4 ◦ C. The supernatant obtained was centrifuged at 105,000 × g for 60 min at 4 ◦ C. The microsomes were washed with ice-cold 0.15 M KCl, and then
stored at −20 ◦ C. The lipid peroxidation assay was performed in Fe2+ /vitamin C system. The microsomes (300 g/mL) were incubated at 37 ◦ C for 60 min with varying concentrations of ascophyllan fractions (10–50 g/mL), 10 mM FeS04 ·7H2 O and 0.1 mM ascorbic acid in 1.0 mL potassium phosphate buffer (0.2 M, pH 7.4). The reaction was stopped by the addition of 20% trichloroacetic acid (1.0 mL) and 0.67% 2-thiobarbituric acid (TBA) (1.5 mL) in succession, and the solution was then heated at 100 ◦ C for 15 min [19]. The condensation reaction occurring between the MDA and TBA produces a pink compound, which has a strong absorption at 532 nm [20]. The percentage of antioxidant activity of the samples was evaluated according to the following formula [21]. Inhibition (rate %) = (A0 − A)/(A0 − Ae ) × 100, where A0 is the absorbance of the free-radical generation system, A is the absorbance of the test sample and Ae is the absorbance of the essential control. 2.4.5. Reducing power assay The reducing power was determined as described by Yen and Chen [22]. Briefly, 0.13 mL of ascophyllan fractions of different concentrations (10–50 g/mL) in phosphate buffer (0.2 M, pH 6.6) were mixed with 0.125 mL of potassium ferricyanide (1%, w/v) and incubated at 50◦ C for 20 min. Afterwards, 0.125 mL of TCA (10%, w/v) were added to the mixture to terminate the reaction. Then, the solution was mixed with 1.5 mL ferric chloride (0.1%, w/v) and the absorbance was measured at 700 nm. 2.5. Statistical analysis The results were analyzed using a statistical program SPSS/PC+, version 11.0 (SPSS Inc., Chicago, IL, USA). Statistical evaluation was done using the one-way ANOVA and significant difference was determined using Duncans test at the level of P < 0.05. 3. Results 3.1. Purification of ascophyllan The total yield of crude ascophyllan (CA) fraction from P. tetrastromatica was 17%. The isolated CA was fractionated by DEAE Sepharose column chromatography using phosphate buffer containing NaCl (0.0–4.0 M) gradient as the eluent. Four fractions namely, ascophyllan fraction1 (AF1) eluted with 0.17 to 0.86 M gradient, ascophyllan fraction 2 (AF2) eluted with 1.25 to 2.05 M gradient, ascophyllan fraction 3 (AF3) eluted with 2.48 to 2.96 M gradient and ascophyllan fraction 4 (AF4) eluted with 3.07 to 3.45 M gradient, respectively. Chemical composition showed that the AF3 fraction contained 42.13% neutral sugars, 15.3% sulphate, 23.5% uronic acid and 0.9% proteins [14]. 3.2. Free-radical-scavenging activity of ascophyllan fractions on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical Fig. 1 shows the DPPH radical-scavenging activity of the ascophyllan fractions from P. tetrastromatica. The scavenging activity of AF1, AF2 and AF4 were 31%, 56% and 16% at 50 g/mL concentration. The AF3 fraction showed an IC50 of 24.7 g/mL while the AF2 fraction showed 39.2 g/mL. The IC50 of AF1 and AF4 was not significant when compared to AF3 and AF2 fractions. However, the IC50 of quercetin, a known free-radical scavenger was only 19.75 ± 1.30 g/mL. 3.3. Superoxide anion scavenging activity of ascophyllan fractions The IC50 values of AF2, AF3 and quercetin were 47.2, 38.9 and 32.3 g/mL; however, the IC50 values of AF1 and AF4 was not much
Please cite this article in press as: Mohsin S, et al. Free-radical-scavenging activity and antioxidant effect of ascophyllan from marine brown algae Padina tetrastromatica. Biomed Prev Nutr (2013), http://dx.doi.org/10.1016/j.bionut.2013.08.006
G Model BIONUT-177; No. of Pages 5
ARTICLE IN PRESS S. Mohsin et al. / Biomedicine & Preventive Nutrition xxx (2013) xxx–xxx
Fig. 1. Scavenging effect of ascophyllan fractions on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. Values are expressed as average of three samples ± SEM.
3
Fig. 3. Scavenging effect of ascophyllan fractions on hydroxyl radicals. Values are expressed as average of three samples ± SEM.
significant. Compared with this result, AF3 showed a stronger scavenging activity for superoxide radicals than other fractions. AF2 and AF3 fraction showed scavenging effect of 53.4% and 75.6% at 50 g/mL concentration. The scavenging effect of the AF1 and AF4 was less than 50%. The results are shown in Fig. 2. 3.4. Hydroxyl radical-scavenging activity of ascophyllan fractions The hydroxyl radical-scavenging effect of ascophyllan fractions and quercetin is shown in Fig. 3. Among the four samples and quercetin, AF3 had stronger scavenging effect against hydroxyl radical. At 50 g/mL, AF3 fraction showed 88.4% scavenging effect while quercetin showed 85.7%. The IC50 values of AF3 and quercetin were 31.2 g/mL and 27.3 g/mL while the IC50 values of AF1, AF2 and AF4 were not much significant. 3.5. Inhibitory effect of ascophyllan fractions on lipid peroxidation
Fig. 4. Inhibition of lipid peroxidation of rat liver microsome by ascophyllan fractions. Values are expressed as average of three samples ± SEM.
3.6. Reducing power of ascophyllan fractions Inhibition of ascophyllan fractions on lipid peroxidation of rat liver microsomes is shown in Fig. 4. The free-radical formation in this system was inhibited by all of the polysaccharide samples with IC50 values of 44.2, 28.2 and 35.2 g/mL for AF2, AF3 and quercetin respectively. At 50 g/mL, AF3 showed 90% inhibition which was similar to quercetin at the same dose.
Fig. 5 depicts the reducing power of tested samples. Higher absorbance value means stronger reducing power of samples. Though the reducing power of AF1 and AF4 was low in the tested concentration, but AF2 and AF3 fractions were concentrationdependent. The reducing power of AF1 and AF4 at 50 g/mL were
Fig. 2. Scavenging effect of ascophyllan fractions on superoxide radicals. Values are expressed as average of three samples ± SEM.
Fig. 5. Reducing power of ascophyllan fractions. Values are expressed as average of three samples ± SEM.
Please cite this article in press as: Mohsin S, et al. Free-radical-scavenging activity and antioxidant effect of ascophyllan from marine brown algae Padina tetrastromatica. Biomed Prev Nutr (2013), http://dx.doi.org/10.1016/j.bionut.2013.08.006
G Model BIONUT-177; No. of Pages 5
ARTICLE IN PRESS S. Mohsin et al. / Biomedicine & Preventive Nutrition xxx (2013) xxx–xxx
4
0.027 and 0.016 respectively, which were much weaker than those of AF2 and AF3. Moreover, the reducing power of quercetin at 50 g/mL was 0.135.
4. Discussion Marine algae are an abundant source of new bioactive natural compounds with a wide range of important biological activities, such as antioxidant, anticoagulant and antithrombotic activities [23]. P. tetrastromatica has been traditionally used in India as a functional food for centuries. In this context, we isolated and characterized a water soluble sulphated polysaccharide from P. tetrastromatica and examined its antioxidant activity. Extraction of algae yielded Crude Ascophyllan (CA), which was approximately 17%. Fractionation of CA by anion exchange column chromatography gave four fractions. 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 [24]. When DPPH free-radical encounters 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 [25] The DPPH radical-scavenging activity of ascophyllan fractions from P. tetrastromatica has been attributed to the ability of the polysaccharide fractions in pairing with the odd electron of DPPH radical [26]. Among them, AF3 fraction showed maximum scavenging activity. Higher radical-scavenging activity was observed in fractions when compared to crude extract [27]. Superoxide radical can be generated by autooxidation and it can produce a coloured compound. Resulting from a colour 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 [28]. The scavenging ability of the ascophyllan fractions decreased in the order of AF3 > AF2 > AF1 > AF4 corresponding to the sulfate content of the samples in the order of 15.3, 6.59, 2.56 and 0.15%, respectively[14]. This clearly demonstrates that the sulfate content affected the antioxidant activity and these results are in agreement with the observations of Qi et al. [29] and Tsiapali et al. [30] who found that sulfated glucan exhibited greater antioxidant ability. Among different reactive oxygen species (ROS), superoxide is generated first [31]. 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 [32]. Our data on the activity of superoxide radical-scavenging suggested that it was likely to contribute towards the observed antioxidant effect. Among the reactive oxygen species, the hydroxyl radical is the most reactive and induces severe damage to adjacent biomolecules. The result suggests that AF3 had the strongest scavenging ability for hydroxyl radicals. Previous studies of the antioxidant activity of various natural plant derived biomolecules have reported that the scavenging activity for hydroxyl radicals is not due to direct scavenging but due to inhibition of hydroxyl radical generation by chelating ions, such as Fe2+ and Cu+ [33]. Lipid peroxidation is a key process in many pathological events and is one of the reactions induced by oxidative stress. The unsaturated fatty acids in cell membrane on oxidation lead to the formation and proliferation of lipid peroxides. The oxygen uptake, structural rearrangements of unsaturated fatty acids and ultimate damage of membrane lipids lead to production of malondialdehyde, which is known to be carcinogenic and mutagenic[34]. The inhibition of lipid peroxidation by AF3 might probably be due to its
free-radical-scavenging potential and thereby proves to be a good antioxidant and cytoprotective agent. It has been reported that reducing power serves as a significant reflection of the antioxidant activity. The presence of reductants in the antioxidant samples cause the reduction of the Fe3+ /ferricyanide complex to the ferrous form. The reducing properties were generally associated with the presence of reductones, which have been shown to exert antioxidant activity by breaking the free-radical chain by donating a hydrogen atom. Most non-enzymatic antioxidative activity, such as scavenging of freeradicals or inhibition of peroxidation, is mediated by redox reaction [35]. Our data shows that reducing power of ascophyllan fractions play a significant role as an antioxidant. 5. Conclusion The results clearly demonstrate that sulfated polysaccharide extracted from P. tetrastromatica was a good antioxidant. AF3 fraction was chosen for further investigation of its antioxidant activities and mechanisms in vivo in our future work. Overall, the present experiments showed that ascophyllan from marine brown algae was a potential therapeutic agent. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgement Financial assistance from UGC in the form of Research Fellowship in Science to Meritorious Students (RFSMS) is gratefully acknowledged. References [1] Blander G, Oliveira RM, Conboy CM, Haigis M, Guarente L. Superoxide dismutase 1 knock-down induces senescence in human fibroblasts. J Biol Chem 2003;278:38966–9. [2] Liu F, Ooi VEC, Chang ST. Free radical scavenging activity of mushroom polysaccharide extracts. Life Sci 1997;60:763–71. [3] Mau J-L, Lin H-C, Chen C-C. Antioxidant properties of several medicinal mushrooms. J Agric Food Chem 2002;50(21):6072–7. [4] Mayer AMS, Lehmann VKB. Marine pharmacology in 1998: marine compounds with antibacterial, anticoagulant, antifungal, anti-inflammatory, anthelmintic, antiplatelet, antiprotozoal and antiviral activities; with actions on the cardiovascular, endocrine, immune and nervous systems: And other miscellaneous mechanisms of action. Pharmacologist 2000;42:62–9. [5] Renn DW. Biotechnology and the red seaweed polysaccharide industry: status, needs and prospects. Trends Biotechnol 1997;15:9–14. [6] Nishino T, Nagumo T. Anticoagulant and antithrombin activities of oversulfated fucans. Carbohydr Res 1992;229:355–62. [7] Koyanagi S, Tanigawa N, Nakagawa H, Soeda S, Shimeno H. Oversulfation of fucoidan enhances its anti-angiogenic and antitumor activities. Biochem Pharmacol 2003;65(2):173–9. [8] Ponce NMA, Pujol CA, Damonte EB, Flores ML, Stortz CA. Fucoidans from the brown seaweed Adenocystis utricularis: extraction methods, antiviral activity and structural studies. Carbohydr Res 2003;338:153–65. [9] Qi H, Zhang Q, Zhao T, Hu R, Zhang K, Li Z. In vitro antioxidant activity of acetylated and benzoylated derivatives of polysaccharide extracted from Ulva pertusa (Chlorophyta). Bioorg Med Chem Lett 2006;16(9):2441–5. [10] Zhang HJ, Mao WJ, Fang F, Li HY, Sun HH, Chen Y, et al. Chemical characteristics and anticoagulant activities of a sulfated polysaccharide and its fragments from Monostroma latissimum. Carbohydr Polym 2008;71:428–34. [11] Lee JB, Hayashi K, Hayashi T, Sankawa U, Maeda M. Antiviral activities against HSV-1, HCMV, and HIV-1 of rhamnan sulfate from Monostroma latissimum. Planta Med 1999;65(5):439–41. [12] Sem SR, Das AK, Siddhanta AK, Mody KH, Ramavat BK, Chauhan VD, et al. A new sulphated polysaccharide with potent anti-coagulant activity from the red seaweed Grateloupia indic. Int J Biol Macromol 1994;16:279–80. [13] Melo MRS, Feitosa JPA, Freitas ALP, De Paula RCM. Isolation and characterisation of sulphated soluble polysaccharide from the red seaweed Gracilaria cornea. Carbohydr Polym 2002;49:491–8. [14] Mohsin S, Muraleedhara Kurup G, Mahadevan R. Effect of ascophyllan from brown algae Padina tetrastromatica on inflammation and
Please cite this article in press as: Mohsin S, et al. Free-radical-scavenging activity and antioxidant effect of ascophyllan from marine brown algae Padina tetrastromatica. Biomed Prev Nutr (2013), http://dx.doi.org/10.1016/j.bionut.2013.08.006
G Model BIONUT-177; No. of Pages 5
ARTICLE IN PRESS S. Mohsin et al. / Biomedicine & Preventive Nutrition xxx (2013) xxx–xxx
[15] [16] [17] [18] [19] [20]
[21]
[22] [23]
[24] [25]
oxidative stress in carrageenan-induced rats. Inflammation 2013, http://dx.doi.org/10.1007/s10753-013-9665-4. Karagozler AA, Erday B, Emek YC, Uggun DA. Antioxidant activity and proline content of leaf extracts from Dorystoechas hastata. Food Chem 2008;111:400–7. Robak J, Gryglewski IR. Flavonoides are scavengers of superoxide anions. Biochem Pharmacol 1988;37:837–41. Smironoff CQ. Hydroxyl radical-scavenging activity of compatible solutes. Phytochemistry 1989;28:1057–60. Liu F, Ooi VCE, Chang ST. Free radical scavenging activities of mushroom polysaccharide extracts. Life Sci 1997;60:763–71. Bueg JA, Aust SD. Microsomal lipid peroxidation. In: Feischer S, Packer L, editors. Methods in Enzymology, 52. New York: Academic Press; 1978. p. 302–10. Wei Z, Bai O, Steven Richardson J, Mousseau DD, Li X. Olanzapine protects PC12 cells from oxidative stress induced by hydrogen peroxide. J Neurosci Res 2003;73:364–8. Zhang Q, Yu P, Li Z, Zhang H, Xu Z, Li P. Antioxidant activities of sulfated polysaccharide fractions from Porphyra haitanesis. J Appl Phycol 2003;15: 305–10. Yen GC, Chen HY. Antioxidant activity of various tea extracts in relation to their antimutagenicity. J Agric Food Chem 1995;43:27–32. Shanmugam M, Mody KH. Heparinoid-active sulphated polysaccharides from marine algae as potential blood anticoagulant agents. Curr Sci 2000;79:1672–83. Huang D, Ou B, Prior RL. The chemistry behind antioxidant capacity assays. J Agric Food Chem 2005;53:1841–56. Li XL, Zhou AG, Li XM. Inhibition of Lycium barbarum polysaccharides and Ganoderma lucidum polysaccharides against oxidative injury induced by irradiation in rat liver mitochondria. Carbohydr Polym 2007;69:172–8.
5
[26] Park PJ, Shahidi F, Jeon YJ. Antioxidant activities of enzymatic extracts from edible seaweed Sargassum horneri using ESR spectroscopy. J Food Lipids 2004;11:15–27. [27] Duan XJ, Zhang W, Li XM, Wang BG. Evaluation of antioxidant property of extract and fractions obtained from a red alga, Polysiphonia urceolata. Food Chem 2006;95:37–43. [28] Chen Y, Xie MY, Nie SP, Li C, Wang YX. Purification, composition analysis and antioxidant activity of a polysaccharide from the fruiting bodies of Ganoderma atrum. Food Chem 2008;107:231–41. [29] Qi H, Zhang Q, Zhao T, Chen R, Zhang H, Niu X, et al. Antioxidant activity of different sulfate content derivatives of polysaccharide extracted from Ulva pertusa (Chlorophyta) in vitro. Int J Biol Macromol 2005;37:195–9. [30] Tsiapali E, Whaley S, Kalbfleisch J, Ensley HE, Browder IW, Williams DL. Glucans exhibit weak antioxidant activity, but stimulate macrophage free radical activity. Free Radic Biol Med 2001;30:393–402. [31] Liu F, Ng TB. Antioxidative and free radical scavenging activities of selected medicinal herbs. Life Sci 2000;66:725–35. [32] Wade CR, Jackson PG, Highton J, van Rij AM. Lipid peroxidation and malondialdehyde in the synovial fluid and plasma of patients with rheumatoid arthritis. Clin Chim Acta 1987;164:245–50. [33] Halliwell B, Gutteridge JMC, Aruoma OI. The deoxyribose method: a simple “test-tube” assay for determination of rate constants for reactions of hydroxyl radicals. Anal Biochem 1987;165:215–9. [34] Miyake T, Shibamoto T. Inhibition of malonaldehyde and acetaldehyde formation from blood plasma oxidation by naturally occurring antioxidants. J Agric Food Chem 1998;46:3694–7. [35] Zhu QY, Hackman RM, Ensunsa JL, Holt RR, Keen CL. Antioxidative activities of oolong tea. J Agric Food Chem 2002;50:6929–34.
Please cite this article in press as: Mohsin S, et al. Free-radical-scavenging activity and antioxidant effect of ascophyllan from marine brown algae Padina tetrastromatica. Biomed Prev Nutr (2013), http://dx.doi.org/10.1016/j.bionut.2013.08.006