Free Radical Scavenging and Antioxidant Enzymes Activation of Polysaccharide Extract from Nostoc sphaeroides

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1 The American Journal of Chinese Medicine, Vol. 35, No. 5, World Scientific Publishing Company Institute for Advanced Research in Asian Science and Medicine Free Radical Scavenging and Antioxidant Enzymes Activation of Polysaccharide Extract from Nostoc sphaeroides Jun Tang, *, Zheng-yu Hu * and Xin-wen Chen * State Key Laboratory of Freshwater Ecology and Biotechnology Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, , China Graduate School of Chinese Academy of Sciences, Beijing, , China State Key Laboratory of Virology, Wuhan Institute of Virology Chinese Academy of Sciences, Wuhan, , China Abstract: Nostoc sphaeroides Kuetzing has been used as a traditional medicine in China to treat a variety of ailments. This research identifies the antioxidant activities of polysaccharide extract from Nostoc sphaeroides. The extract, which contains 46.2% carbohydrates, exhibited an effective scavenging capability on superoxide radical, hydroxyl radicals in non site-specific as well as site-specific assays, and also performed lipid peroxidation in a dose-dependent manner. Polysaccharide extract had no 1,1-diphenyl-2-picrylhydrazyl radical scavenging potential at all test concentrations. Activities of superoxide dismutase, catalase, and glutathione peroxidase in human embryo kidney 293 cells were increased effectively when Nostoc sphaeroides extract was applied. These result suggested that the use of N. sphaeroides in treating ailments may be based on the antioxidant capacities of polysaccharide composition. Keywords: Nostoc sphaeroides Kuetzing; Polysaccharide; Antioxidant activity; Scavenging Effect; Lipid Peroxidation; Human Embryo Kidney 293 Cells. Introduction Reactive oxygen species (ROS) are involved in a number of diseases and degenerative conditions (Wiseman and Halliwell, 1996; Finkel and Holbrook, 2000). To protect themselves from oxidative damages, cells have developed a variety of antioxidant defense mechanisms. These include antioxidant enzymes such as superoxide dismutase (SOD), Correspondence to: Dr. Zheng-yu Hu, Institute of Hydrobiology, Chinese Academy of Sciences, 7 South Donghu Road, Wuchang District, Wuhan, Hubei Province, , China. Tel: (+86) ; Fax: (+86) , Huzy@ihb.ac.cn indd 887 9/13/2007 7:35:10 PM

2 888 J. TANG et al. catalase (CAT) and glutathione peroxidase (GPX), as well as some small antioxidant molecules. ROS can cause extensive damages to DNA, proteins and other biomolecules when the production of radicals grossly exceeds the antioxidant capacity of the cell or organism (Lee et al., 2003; Sun et al., 2004). Numerous studies have proven that antioxidants from natural resources could provide further protection against oxidative stress (Schinella et al., 2002; Di Matteo and Esposito, 2003). Nostoc sphaeroides, a nitrogen-fixing cyanobacterium, grows in mountain paddy fields of China in the winter in the form of deep green spherical colonies. It has been used as food delicacy and folk herbal medicine to treat nyctalopia, burn and some other ailments since the Eastern Jin Dynasty ( AD), as recorded in The Supplement to Compendium of Materia Medica (Qiu and Liu, 2004; Gao and Ai, 2004). Nevertheless, very little research has been done on the bioactivities of N. sphaeroides polysaccharide extract, in particular as it relates to potential therapeutical effects. In the present study, antioxidant activities of N. sphaeroides polysaccharide extract were assessed by their scavenging abilities on free radicals. The effect of the extract on SOD, CAT and GPX in human embryo kidney 293 cells was also investigated. Material and Methods Chemicals Phenazine methosulphate (PMS), nitro blue tetrazolium (NBT), 2-deoxy-D-ribose (DR), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), ferrozine, nicotinamide adenine dinucleotide-reduced (NADH), dimethylsulphoxide (DMSO) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich Co. D-mannitol and L-ascorbic acid were purchased from Duchefa, Netherlands. Tri (hydroxymethyl) aminomethane (Tris-HCl) was purchased from Promega. SOD, CAT and GPX kits were obtained from the Institute of Nanjing Jiancheng Biological Engineering (China). All other reagents were of analytical grade. Preparation of N. sphaeroides Polysaccharide Extract N. sphaeroides was cultured in an axenic BG-11 medium. The dried ground materials (10 mg) were suspended in ethanol (80% v/v) overnight and then extracted under reflux until the solvent fraction was colorless. The powder was suspended in distilled water overnight followed by boiling at 95ºC for 3 hours. The residue was extracted for another 3 hours at 95ºC. The supernatant was pooled and concentrated in a rotary evaporator. Ethanol was added to the concentrated extract at a ratio of 3 to 1. The precipitate was collected by centrifugation and washed twice sequentially with ethanol and acetone. After being dissolved in distilled water, the extract was filtered, lyophilized and stored at 4ºC indd 888 9/13/2007 7:35:10 PM

3 ANTIOXIDANT ENZYMES ACTIVATION OF NOSTOC SPHAEROIDES 889 Superoxide Anion Radical Scavenging Assay The scavenging effect on superoxide anion radicals was assessed using the NBT reduction method (Nishikimi et al., 1972) with some modifications. Briefly, the superoxide radical was generated in 4 ml of Tris-HCl buffer (20 mm ph 8.0), containing PMS (20 µm), NADH (100 µm), NBT (50 µm) and the extract at various concentrations (2, 6, 12, 24 and 40 µg/ml). The reaction mixture was shaken well and incubated for 5 min at ambient temperature. Absorbance of the resulting mixture was read at 560 nm against a blank. The percentage inhibition values were calculated following the equation: Scavenging effect (%) = (1 A s /A c ) 100, where A c is the absorbance of control, and A s is that of the sample. Non Site-Specific and Site-Specific Hydroxyl Radical Scavenging Assays The non site-specific hydroxyl radical scavenging effect was investigated using the deoxyribose method (Halliwell et al., 1987) with slight modifications. The reaction mixture, containing EDTA (104 µm), FeCl 3 6H 2 O (100 µm), ascorbate acid (100 µm), 2-deoxy-ribose (2.8 mm), hydrogen peroxide (1 mm) and the polysaccharide extract at various concentrations (6, 12, 18 and 30 µg/ml)in potassium phosphate buffer (20 mm ph 7.4) was incubated at 37ºC for 60 min. Equal volumes of trichloroacetic acid (TCA, 10% w/v) and TBA (1% w/v) were added to terminate the reaction. The mixture was then boiled for 15 min and the absorbance was measured at 532 nm against a blank. D-mannitol, an effective hydroxyl radical scavenger, was used as a positive control. The scavenging effect was calculated as follows: Scavenging effect (%) = (1 A s /A c ) 100, where A c is the absorbance of control, and A s is that of the sample. The site-specific hydroxyl radical scavenging assay was performed as described above except that EDTA was replaced by an equivalent volume of phosphate buffer (Sun et al., 2004). DPPH Radical Scavenging Assay The DPPH free radical scavenging assay was carried out following the procedure described by Yamaguchi et al. (1998), with a slight modification. DPPH (100 µm) solution was added to the sample at various concentrations (2, 6, 12, 24 and 36 µg/ml) and the volume was adjusted to 3 ml by adding ethanol (80%). The reaction mixture was shaken vigorously and allowed to stand at room temperature for 30 min. The scavenging capacity was measured by monitoring the decrease in absorbance at 517 nm. Lipid Peroxidation Inhibition in Egg Yolk Homogenate The inhibition effect of the N. sphaeroides extract on lipid peroxidation, based on the formation of malondialdehyde (MDA), was investigated employing the modified thiobarbituric acid reactive substances (TBARS) assay (Ohkowa et al., 1979), using egg yolk homogenate as lipid rich media (Dorman et al., 1995; Ruberto et al., 2000). Egg indd 889 9/13/2007 7:35:10 PM

4 890 J. TANG et al. homogenates (10% in 1.15% KCl, v/v) were mixed with FeSO 4 (100 µm), H 2 O 2 (50 µm) and extract (10, 20, 40 and 100 µg/ml). The mixture was filled to 1 ml with distilled water and incubated at 37ºC for 1 hour, followed by the addition of 1.5 ml of TCA (20%, ph 3.5) and 1.5 ml of TBA (0.67%, w/v). The reagent was boiled for 15 min. After centrifuged at 3,000 rpm for 15 min, the absorbance of the supernatant was monitored at 532 nm (Janero, 1990). The inhibition rate was calculated according to the following equation: % inhibition = (1 A s /A c ) 100, where A c is the absorbance of control, and A s is that of the sample. Cell Culture Human embryo kidney 293 cells were cultured in Dalbecco s modified Eagle s medium (DMEM) containing 10% fetal bovine serum and maintained at 37ºC in a humidified atmosphere of 5% CO 2 and 95% O 2. Cell Viability Cell viability was determined by the modified MTT assay, which is based on the cleavage of a tetrazolium salt by mitochondrial dehydrogenase in viable cells (Hansen et al., 1989). Cells were seeded in 96 well plates at a density of cells per well. After 24 hours incubation, cells were treated with fresh medium containing various concentrations of N. sphaeroides extract ( µg/ml). Incubation was continued for another 24 hours, 48 hours and 72 hours. During the last 4 hours, MTT stock solution (5 mg/ml) was added. The MTT solutions were drained off and 100 ìl of DMSO was added to dissolve the formazan crystals for 15 min. The absorbance was measured using an ELISA reader (Bio-Rad, USA) at 570 nm. Data are mean percentages of viable cells of 6 replications versus respective controls. The optical density of the formazan formed in the control groups was designated as 100% viability. Antioxidant Enzyme Assays Cells were collected and suspended in phosphate buffer (20 mm, ph 7.4) after treating with 1, 5 and 25 µg/ml of extract for 24 hours. Then cells were homogenized by sonication and centrifugated at 14,000 g for 15 min. The supernatant was collected. Protein concentrations were quantified by Bradford s method (Bradford, 1976). Bovine serum albumin was used as a standard. Activities of SOD, CAT and GPX were measured using enzyme kits. Total Carbohydrate Content Total carbohydrate content was evaluated according to the phenol-sulfuric acid method (Dubois et al., 1956). Samples were incubated with phenol solution (6%) and concentrated indd 890 9/13/2007 7:35:10 PM

5 ANTIOXIDANT ENZYMES ACTIVATION OF NOSTOC SPHAEROIDES 891 sulfuric acid for 15 min at room temperature. The absorbance was monitored at 490 nm. The amount of carbohydrates was calculated with reference to a standard curve using glucose as a standard. Statistical Analysis All data are expressed as mean ± SD. One-way analysis of variance (ANOVA, Origin 7.0) was used for data process. p < 0.05 were regarded as significantly different. Results and Discussion Superoxide Radical Scavenging Activity Superoxide radicals can cause severe damage to cells and tissues through forming other reactive oxygen species such as singlet oxygen and hydroxyl radicals (Dahl and Richardson, 1978). As shown in Fig. 1, the extract exhibited significant scavenging effects at all tested concentrations. The effects obviously increased with increasing sample concentrations up to a certain extent (6 µg/ml), followed by a relatively smaller increase ( %) with further increase in concentrations (6 40 µg/ml). Non Site-Specific and Site-Specific Hydroxyl Radical Scavenging Activity Among the reactive oxygen species, hydroxyl radicals are considered to be the most reactive, because it can react with all biomacromolecules functioning in living cells. In particular, it can initiate membrane lipid peroxidation (Liu and Ng, 2000). The sample showed both non site-specific and site-specific hydroxyl radical scavenging activities in a concentration-dependent fashion (Fig. 2). The suppressive effect of the extract on deoxyribose degradation was much higher in site-specific assay than in non site-specific one at each concentration tested, with IC 20 values (concentration of sample required to scavenge 20% of free radicals or to prevent lipid peroxidation by 20%) of 26.8 µg/ml and 34.7 µg/ml, respectively. These numbers were much lower than that of D-mannitol (11.2 µg/ml). In the site-specific assay, the extract was assumed to be able to prevent deoxyribose damage associated with the direct binding of iron to deoxyribose and the subsequent attack by OH. While in non site-specific protocol, the ability was speculated to be the direct scavenging on OH, for the chelation between Fe 2+ and polysaccharide extract was diminished by EDTA. And accordingly the formation of OH was predominant in the case. DPPH Radical Scavenging Capacity DPPH is a stable free radical and exhibits a strong purple color in methanol solution with a characteristic absorption at 517 nm. Antioxidants neutralize the free radical character of indd 891 9/13/2007 7:35:11 PM

6 892 J. TANG et al. Figure 1. Superoxide radical scavenging activity of N. sphaeroides extract. Values were presented as mean ± SD (n = 3). Figure 2. Scavenging effects of N. sphaeroides extract on hydroxyl radical in non site-specific and site-specific assays. D-mannitol was used as a positive control. Values were presented as mean ± SD (n = 3). DPPH by transferring electrons or donating hydrogen atoms and causing the color of the reaction mixture to change from purple to yellow (Naik et al., 2003; Banerjee et al., 2005). The degree of the discoloration indicates the scavenging potential of the antioxidants. The extract showed no quenching effect on DPPH radical (data not shown). The water soluble extract from N. sphaeroides could barely dissolve in ethanol, and this may limit the quenching ability. Besides the solubility, stereo selectivity of radicals and structure of the extract compound should be taken into account as well indd 892 9/13/2007 7:35:11 PM

7 ANTIOXIDANT ENZYMES ACTIVATION OF NOSTOC SPHAEROIDES 893 Lipid Peroxidation Inhibition Membrane lipids are susceptible to oxidation, not only because of their high polyunsaturated fatty acid content but also their association in the cell membrane with enzymatic and non-enzymatic systems capable of generating free radical species. Lipid peroxidation is characteristically a free radical chain reaction triggered by the abstraction of a hydrogen atom from a polyunsaturated fatty acid side chain. This was found to be an important cause of cell membrane destruction and cell damage. Therefore, inhibition of lipid peroxidation has been used as an important index of antioxidant activity. Figure 3 depicts that the extract had a significant inhibition effect on lipid peroxidation with an IC 20 value of 31.8 µg/ml and the ability was steadily increased with the increasing concentrations of the extract. Antioxidant Enzyme Assay in Human Embryo Kidney 293 Cells Figure 4 shows that there was no cytotoxicity in the groups treated with N. sphaeroides extract in the range from 0.5 to 30 µg/ml. Extract significantly activated SOD and CAT levels by 11.1 and 29.4% at 5 µg/ml and by 29.0 and 61.1% at 25 µg/ml, respectively. GPX activity was significantly increased at each test concentration from 6.1 to 56.4% (Table 1). SOD, CAT and GPX are three very important antioxidant enzymes in the protection of cells against oxidation. Generally, they are involved in the modulation of the balance between the generation and scavenging of free radicals, thus maintaining the biological homeostasis. SOD catalyzes the dismutation of superoxide oxygen to hydrogen peroxide, which is further scavenged by CAT and GPX. Polysaccharide extract of N. sphaeroides effectively enhanced the activities of these three enzymes. Among which, CAT was activated to a higher extent than GPX and SOD. This may indicate that CAT is more important in the enzyme defense systems in human embryo kidney 293 cells. Figure 3. Inhibition of lipid peroxidation of N. sphaeroides extract in egg yolk homogenate. Values were presented as mean ± SD (n = 3) indd 893 9/13/2007 7:35:11 PM

8 894 J. TANG et al. Figure 4. MTT assay of N. sphaeroides on human embryonic kidney 293 cells. Values were presented as mean ± SD (n = 6). a The optical density of the formazan formed in the control groups was designated as 100% viability. Table 1. Assay of SOD, CAT and GPX Activities in Human Embryo Kidney 293 Cells after 24 hours Extract Treatment Concentration of SOD (U/mgprot) CAT (U/gprot) GPX (µmol/min/mg) Extract (µg/ml) Control Extract Control Extract Control Extract ± ± ± ± ± ± 0.2 * ± ± 1.3 * 47.5 ± ± 0.6 * 11.9 ± ± 0.3 * ± ± 21.4 * 33.2 ± ± 0.6 * 10.6 ± ± 0.6 * a Values were presented as mean ± SD (n = 3). * p < 0.05 versus the control. Total Carbohydrate Contents Total carbohydrate content of N. sphaeroides extract was 46.2% (y = x , R 2 = ). Polysaccharides have been proven to be a promising natural source of antioxidants. They possess many favorable biological properties. The antioxidant abilities demonstrated in this study may partially be ascribed to the polysaccharides contained in the extract. Compositionally, the extract consists of rhamnose, fucose, xylose, mannose, galactose, and glucose (Huang et al., 1998). These compounds are actually potent reductive agents. This may further indicate that the polysaccharide components contribute in part to the antioxidant capacities of the extract. In conclusion, the results obtained in the present study clearly demonstrate that the polysaccharide extract of N. sphaeroides has excellent antioxidant abilities. It can offer effective protection against oxidative stress. This suggests that the use of N. sphaeroides in treating ailments may be based on the antioxidant effect of its polysaccharide constituent indd 894 9/13/2007 7:35:12 PM

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