Hydroxyl radical scavenging activity

1. Iron-EDTA solution: 13mg of ammonium ferrous sulphate and 260mg of EDTA in 100ml of distilled water. 2. Ascorbic acid: 220mg in 100ml distilled water. 3. DMSO solution: 1.70ml dimethyl sulphoxide in 200ml of phosphate buffer (PH 7.4). 4. Trichloacetic acid (17.5% w/v). 5. EDTA (Ethylene Diamine Tetra Acetic) solution (0.01M): 18mg of EDTA was dissolved in 100ml of distilled water. 6. Nash reagent: 150g of ammonium acetate, 3ml of glacial acetic acid and 2ml of acetyl acetone were mixed and raised to 1 liter with distilled water. 7. Test solution: 1mg/ml solution of all the extracts of S. mahagoni seed was prepared in methanol. 8. Hydroxyl radical scavenging activity was measured according to (Singh et al., 2002). Various concentrations 10, 50, 100 and 250 and 500g/ml of samples were taken in different test tubes and volume was made up to 250l with 0.1M phosphate buffer. 1 ml of iron-EDTA solution (0.13% ferrous ammonium sulfate and 0.26% EDTA), 0.5 ml of EDTA (0.018%), and 1 ml of Dimethyl sulphoxide (0.85% v/v in 0.1 M phosphate buffer, pH 7.4) were added to these tubes, and the reaction was initiated by adding 0.5 ml of 0.22% ascorbic acid. These reaction mixtures were incubated at room temperature for 15 min. The reaction was terminated by the addition of 1 ml ice-cold TCA (17.5% w/v). 3 ml of Nash reagent (150 g of ammonium acetate, 3 ml of glacial acetic acid and 2 ml of acetyl acetone were mixed and made up to 1 lit.

with distilled water) was added to each tubes and left at room temperature for 15 min. The intensity of the yellow color formed was measured spectrophotometrically at 412 nm against reagent blank. The percentage of hydroxyl radical scavenging activity was calculated by the formula: 9.
( )

Reagents: 1. Phosphate buffered saline (pH 7.0): 8 gm of Nacl; 1.21 gm K2HPO4 + 0.34 gm KH2PO4 in one litre of distilled water. 2. Sodium nitroprusside (10 mM) solution: 29.79 mg of Sodium nitroprusside in 10 ml of phosphate buffered saline (pH 7.0). 3. Griess reagent: Solution A: 1 gm sulphanilamide dissolved in 5% H3PO4 solution (5 ml of H3PO4 in 100 ml de-ionized water). Solution B: 0.1% N- napthyl ethylene diamine (10 mg in 10 ml de-ionized water). 4. Test solution: Ethanolic, methanolic and aqueous extracts of S. mahagoni seed. Procedure: Various concentrations 10, 50, 100 and 250, 500, 750 and 1000g of extracts were taken in different test tubes and made up to 3ml with 0.1M phosphate buffer (pH 7.2). 1 ml Sodium Nitroprusside (5mM prepared in buffered saline pH7.2) was added to each tube. The reaction mixture was incubated for 30 min at room temperature. After 30 min, 1.5 ml of above solution was mixed with 1.5 ml of Griess reagent (1% Sulphanilamide, 2% phosphoric acid and 0.1% N-1Naphthylethylenediamine dihydro chloride). Control without the test compound, but with an equivalent amount of methanol was maintained. The

absorbance of the samples was measured at 546 nm (Kumar et al, 2008). Nitric oxide radical scavenging activity was calculated using the formula: ( )

Where OD control= absorbance of control, sample OD= absorbance of the extract. 2.6.6. Ferric reducing power activity Measurements of the reducing ability, the Fe3+Fe2+ transformation was investigated in the presence of S. mahagoni seeds oil. The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity (Yildirim et al., 2000). With the increase of reducing power, the chemical conversion will be enhanced and the concentration of the respective chromophores formed will be proportional accordingly. Increased absorbance of the reaction mixture/chromophores formed indicates the increase in reducing power. The basic mechanism of action of almost all chemical antioxidants is reducing reaction. Hence, an increase in reducing power with concentration is an indication of antioxidant potential of the plant extracts. Reagents: 1. Phosphate buffer (0.2M. PH 6.6): NaH2PO4- 17.885% + Na2HPO4-1.8868%. 2. K4Fe(CN)6: 1% 3. Trichloro acetic acid: 10% 4. Fresh Fecl3: 0.1% 5. Test solutions: Ethanolic, methanolic and aqueous extracts of S mahagoni seed. Procedure Ferric reducing scavenging activity was determined by Barreira et al., (2008). Various concentrations of sample 10, 50, 100, 250, 500, 750 and 1000g were mixed with

2.5 ml of 200 mM sodium phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide. The mixture was incubated at 50C for 20 min; then 2.5 ml of 10% (w/v) trichloroacetic acid was added. 5 ml of above solution was mixed with 5 ml of distilled water and 1 ml of 0.1% of ferric chloride. The absorbance was measured spectrophotometrically at 700 nm. BHA was used as standard antioxidant. 1.6.2. DPPH (1, 1-diphenyl-2-picrylhydrazyl) free radical scavenging activity The free radical-scavenging activity of the extract was measured in terms of hydrogen donating or radical-scavenging ability using the stable radical DPPH (Singh et al., 2002). DPPH is a stable free radical containing an odd electron in its structure and usually utilized for detection of the radical scavenging activity in chemical analysis. DPPH is a stable free radical at room temperature and accepts an electron or hydrogen radical to become a stable diamagnetic molecule (Soares et al., 1997) that readily undergo reduction by antioxidant substances at 517nm. The scavenging activity is measured from the discoloration of DPPH. Due to the availability, reproducibility and convenience, it has become a routine assay in studying antioxidant.

Reagents: 1. DPPH solution: A working solution of ethanolic DPPH was used. This was prepared by taking 150 l of stock solution (50 mg of DPPH IN 25 ml of ethanol) in 3 ml of ethanol. 2. Test solution: Ethanolic, methanolic and aqueous extracts of S. mahagoni seed. Procedure

Different concentrations (10, 50,100, 250 and 500g/ml of extract and reference drug BHA were mixed with 5 ml of methanolic solution of DPPH (0.1 mM). The test solutions were allowed to stand at room temperature for 20 min. The absorbance of the samples was measured at 517 nm. Reagent solution without test samples was used as control. Radical scavenging activity was calculated by using the formula: ( )