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Streptomyces species being the largest members of actinomycetes have been known for their metabolic capacities and form an august source of pharmacologically important compounds. The present study concentrated on in vitro evaluation of antioxidant property of n-butanol extract with antimicrobial potential from Streptomyces fradiae strain GOS1. The antioxidant activity was evaluated by total phenolic content, total reductive capability, DPPH assay, lipid peroxidation inhibition assay, nitric oxide scavenging activity, super oxide anion scavenging activity and metal chelating activity. The result showed moderate dose dependent antioxidant activity of the metabolite and is indicative of its role as a primary antioxidant. *Corresponding author, Mailing address: Onkarappa R* E-Mail: *[email protected], [email protected] Article History:-----------------------Date of Submission: 03-01-2013 Date of Acceptance: 28-01-2013 Conflict of Interest: NIL Source of Support: NONE F u ll L e n g t h R e s e a r c h M a n u s c r ip t Int. J. Drug Dev. & Res., January-March 2013, 5 (1): 235-244 Covered in Scopus & Embase, Elsevier 235 These mechanisms are not sufficient when the balance shifts to the side of free radical generation, thus body requires antioxidant supplements to reduce oxidative damages [5]. Antioxidants are molecules capable of preventing oxidative damage. Recent investigations suggest that antioxidant capacity of putative antioxidants can be attributed to various mechanisms such as prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction and radical scavenging in body cells alleviating lipid, protein oxidation and may reduce potential mutations. Therefore help prevent degenerative diseases and other pathologies [6,7]. The wide use of natural antioxidants as a replacement of conventional synthetic antioxidants in food and food supplements has been employed owing to the fact that natural products are considered to be a promising and safe source. Microbial natural products are notable not only for their potent therapeutic activities, but also for the fact that they frequently possess the desirable pharmacokinetic properties required for clinical development; many microbial natural products reach market without any chemical modifications, a testimony to the remarkable ability of microorganisms to produce drug like small molecules[8]. Among microorganisms the members of the order Actinomycetales have proved to be a particularly rich source of secondary metabolites with extensive industrial applications [9]. Traditionally actinomycetes have been the most prolific group in antibiotic production and have been the source of a good number of marketed antibiotics [10]. Mathematical models have suggested that the number of compounds still to be discovered from actinomycetes could well be above 105 a tiny fraction of which has been unearthed so far [11]. The genus Streptomyces represents the largest group of actinomycetes and is the largest antibioticproducing genus in the microbial world discovered so far. The number of antimicrobial compounds reported from the species of this genus per year increased almost exponentially [11]. Streptomycetes are used extensively in industry, due to their ability to generate a number of chemical compounds, including antibiotics, pigments, enzymes, enzyme inhibitors, antitumour agents, antifungal compounds, antiviral agents, pesticides etc[12, 13, 14]. The capacity of the members of this genus to produce commercially significant compounds, especially antibiotics, remains unsurpassed [15]. In pretext of the need of antioxidants and the rich and diverse secondary metabolic capacity of Streptomyces, the present study was carried out to assess the antioxidant potential of a bioactive fraction from Streptomyces fradiae strain GOS1 isolated from Western Ghats soils of Agumbe, Karnataka, India. S. fradiae strain GOS1 is a gram positive, non-acid fast, broad spectrum antibiotic producing, grey coloured isolate with an extended spiral spore chain arrangement and smooth spore surface arrangement (Figure 1)[16]. (a) (b) (c) Figure 1: Colony and Microscopic characteristics of Streptomyces fradiae GOS1 [aColony on Starch casein agar; bSpore arrangement (2000x), cSEM of spore chain (10000x)] F u ll L e n g t h R e s e a r c h M a n u s c r ip t Onkarappa R et al: In Vitro Antioxidant activity of Metabolite from Streptomyces fradiae strain GOS1 Int. J. Drug Dev. & Res., January-March 2013, 5 (1): 235-244 Covered in Scopus & Embase, Elsevier 236 MATERIALS AND METHODS: Chemicals and Reagents: Chemicals, such as Ascorbic acid, Quercetin, Gallic acid, 1, 1 Diphenyl-2-picrylhydrazyl (DPPH), NADH, Nitro Blue Tetrazolium (NBT), Catechin were procured from Sigma Chemical Co. (St Louis, MO, USA), Ferrozine, Ferrous Chloride, Folin-Ciocalteu reagent was from Spectrochem Pvt. Ltd. All other chemicals unless and otherwise mentioned were obtained from Sisco Research Laboratories Pvt. Ltd. (Mumbai, India). Fermentation: The isolate was grown in 250ml Erlenmeyer flasks containing 150ml of Starch Casein Nitrate (SCN) broth. The inoculated flasks were incubated at 30±2°C for 12 days. The growth was constantly observed for any possible contaminations and the broth was filtered through Whatmann Grade-01 filter paper[17]. Solubility and Metabolite Extraction: The culture filterate was subjected to solubility tests in different solvents viz., hexane, n-butanol, ethyl acetate, acetone, methanol and ethanol on polarity basis and LD50 values[18]. The solubility assessment was done by mixing broth and solvent (1:1v/v). The metabolite was found to be soluble in n-butanol. The extraction was carried out using sterile separating funnel to which equal volumes (1:1v/v) of broth and solvent were added and was vigorously agitated to ensure proper mixing of solvent and broth. The funnel was allowed to stand for 30 min. The organic fraction was separated, dried and used for antioxidant assays [19,20]. Antioxidant Activity: DPPH Radical Scavenging Activity: The free radical scavenging activity of the metabolite, based on the scavenging activity of the stable 1,1diphenyl-2-picrylhydrazyl (DPPH) free radical was assayed according to the protocol of Braca et al.[21]. 0.1ml of n-butanol extract (0-100μg/ml of methanol) was added to 3ml of 0.004% DPPH (in methanol) and the tubes were incubated in dark at room temperature for 30 min and the absorbance was measured at 517nm spectrophotometrically. Tertiary butyl hydroquinone (TBHQ) was used as standard. Absorbance of DPPH without extract/standard was also noted. The DPPH radical scavenging activity (%) was calculated using the formula, Scavenging activity (%) = [(Ao – Ae) / Ao] × 100 Where Ao is the absorbance of the control and Ae is the absorbance of the metabolite/standard. Total Reductive Capability: 1ml of different concentrations (0-100μg/ml) of nbutanol extract and the standard (Ascorbic acid) was mixed with 2.5ml of 0.2M phosphate buffer (pH 6.6) and 2.5ml of 1% potassium ferricyanide [K3Fe(CN)6]. Reaction mixture was incubated at 50°C for 20 min, cooled to room temperature, 2.5ml of 10% trichloro acetic acid (TCA) was added and centrifuged (6500rpm at room temperature) for 10min. The upper layer of solution (2.5ml) was mixed with 2.5ml of distilled water and 0.5ml of 0.1% Ferric Chloride. Absorbance of the reaction mixture was measured at 700nm in a spectrophotometer. Increased absorbance of the reaction mixture indicates the increase in reduction capability[22]. Metal Chelating Activity: The chelating of ferrous ions by n-butanol extract and standard ethylene diamine tetra acetic acid (EDTA) was estimated according to the method of Dinis et al.[23]. The extract and standard (0200μg/ml) were mixed with distilled water to make the volume to 3.0ml and 2mM 0.05ml of ferrous chloride (FeCl2) solution was added. The reaction was initiated by the addition of 5mM ferrozine (0.2ml) and the mixture was vortexed and was incubated at room temperature for 10min. Absorbance of the solution was measured spectrophotometrically at 562nm. The control contained ferrous chloride and ferrozine complex formation molecules. The percentage of inhibition of Ferrozine-Fe2+ complex formation was calculated from the formula, F u ll L e n g t h R e s e a r c h M a n u s c r ip t Onkarappa R et al: In Vitro Antioxidant activity of Metabolite from Streptomyces fradiae strain GOS1 Int. J. Drug Dev. & Res., January-March 2013, 5 (1): 235-244 Covered in Scopus & Embase, Elsevier 237 Inhibition (%) = [(Ao – Ae) / Ao] × 100 Where Ao is the absorbance of the control and Ae is the absorbance of the metabolite/standard. Lipid Peroxidation Inhibition Assay: A modified thiobarbituric acid reactive species (TBARS) assay was used to measure lipid peroxidation formed using egg yolk homogenate as lipid rich media. 0.5ml of egg homogenate (10% in distilled water) and 0.1ml of extract and standard Butylated Hydroxyanisole (BHA) in the concentration range 0-125μg/ml were mixed in test tubes, the volume was made up to 1ml by adding distilled water. Finally 0.05ml of 0.07M ferrous sulphate (FeSO4) was added to the mixture and incubated for 30min to induce the lipid peroxidation. Further 1.5ml of 20% acetic acid (pH 3.5), 1.5ml of 0.8% TBA and 0.05ml 20% TCA was added, the mixture was vortexed and was heated in a boiling water bath for 60min. After cooling 5ml of n-butanol was added and centrifuged at 3000rpm for 10min. The absorbance of the organic upper layer was measured at 532nm using a spectrophotometer[24]. The percentage inhibition of lipid peroxidation was calculated from the formula, Inhibition (%) = [(Ao – Ae) / Ao] × 100 Where Ao is the absorbance of the control and Ae is the absorbance of the extract/standard. Superoxide Radical Scavenging Activity: The superoxide radical scavenging activity was assayed according to the method of Nishimiki et al.[25]. Superoxide anions were generated in a nonenzymatic phenazine methosulfate-nicotinamide adenine dinucleotide (PMS-NADH) system through the reaction of PMS-NADH and oxygen. It was assayed by the reduction of nitroblue tetrazolium (NBT). All the solutions used in this experiment were prepared in phosphate buffer (pH 7.4). 1ml of NBT (156μM), 1ml of NADH (468μM) and 1ml of extract (000-125μg/ml) were mixed. The reaction was started by adding 1ml of PMS (60μM) and the mixture was incubated at 25°C for 5min followed by measurement of absorbance at 560nm spectrophotometrically. Decreased absorbance of the reaction mixture indicated increased superoxide anion scavenging activity. Ascorbic acid was used as standard. The percentage inhibition was calculated from the formula, Inhibition (%) = [(Ao – Ae) / Ao] × 100 Where Ao is the absorbance of the control and Ae is the absorbance of the extract/standard. Nitric Oxide Radical Scavenging Activity: Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide, which interacts with oxygen to produce nitrite ions, which can be determined by the use of the Griess Illosvoy reaction. 2ml of 10mM sodium nitroprusside in 0.5ml of phosphate buffer saline (pH 7.4) was mixed with 0.5ml of extract and standard (curcumin) at various concentrations and the mixture was incubated at 25°C for 150min. From the incubated mixture 0.5ml was taken out and added into 1ml of sulfanilic acid reagent and incubated for 5min. Finally 1ml napthylethylenediamine dihydrochloride (0.1% w/v) was mixed and incubated at room temperature for 30min before measuring absorbance at 540nm using spectrophotometer[26]. Nitric oxide radical scavenging activity was calculated using the formula, Inhibition (%) = [(Ao – Ae) / Ao] × 100 Where Ao is the absorbance of the control and Ae is the absorbance of the extract/standard. Total Phenolic Content: The total phenolic content (TPC) of the extract was determined with the Folin-Ciocalteu reagent (FCR). 1.0ml of extract was mixed with 2.5ml FCR (diluted 1:10v/v) followed by 2ml of sodium carbonate (7.5% v/v) solution. The tubes were vortexed and allowed to stand for 90min at room temperature. Absorbance was measured against the blank at 750nm using a spectrophotometer. The TPC of the extract was expressed in terms of milligrams (mg) of gallic acid equivalents (GAE)/g of dry weight [27]. F u ll L e n g t h R e s e a r c h M a n u s c r ip t Onkarappa R et al: In Vitro Antioxidant activity of Metabolite from Streptomyces fradiae strain GOS1 Int. J. Drug Dev. & Res., January-March 2013, 5 (1): 235-244 Covered in Scopus & Embase, Elsevier 238 RESULTS: DPPH Radical Scavenging Activity: The scavenging of DPPH free radicals was observed by decrease in absorbance at 517nm induced by extract/standard. The results showed dose dependent scavenging activity. The percentage of inhibition of DPPH radicals by n-butanol extract and TBHQ at concentration of 100μg/ml was 33.33% and 68.4% respectively (Figure 2). Figure 2: DPPH radical scavenging activity of nbutanol extract and TBHQ Total Reductive Capability: In total reductive assay, the increase in absorbance at 700nm with the increase in concentration showed the reducing capability of standard and the n-butanol extract. Ascorbic acid showed absorbance values of 1.15 and the n-butanol extract showed 0.51 absorbance units at concentrations of 200μg/ml respectively (Figure 3). Figure 3: Total reductive capability of n-butanol extract and ascorbic acid Metal Chelating Activity: Metal chelating capacity of the extract was dose dependent and was evident by the decrease in absorbance at 562nm. EDTA showed higher chelating efficacy than extract. The percentage of metal chelating capacity at 200μg/ml of EDTA and nbutanol extract was found to be 70.48% and 39.54% respectively (Figure 4). Figure 4: Metal chelating activity of n-butanol extract and EDTA Lipid Peroxidation Inhibition Assay: In lipid peroxidation inhibiton assay, n-butanol extract and BHA showed a dose dependent activity and the inhibition increased with the increase in concentration. The percentages of lipid peroxidation inhibition capacity of BHA and extract at 125μg/ml were found to be 90.93% and 68.13% respectively (Figure 5). Figure 5: Lipid peroxidation inhibition by n-butanol extract and BHA Superoxide Radical Scavenging Activity: The percentage of superoxide scavenging activity by the n-butanol extract and the standard was dose dependent. The percentage radical scavenging activity of ascorbic acid and n-butanol extract at F u ll L e n g t h R e s e a r c h M a n u s c r ip t Onkarappa R et al: In Vitro Antioxidant activity of Metabolite from Streptomyces fradiae strain GOS1 Int. J. Drug Dev. & Res., January-March 2013, 5 (1): 235-244 Covered in Scopus & Embase, Elsevier 239 125μg/ml was 69.25% and 48.28% respectively