Where A518 control is the absorbance of DPPH radical+ methanol; A518 sample is the absorbance of DPPH radical + extract or compound / standard.
Superoxide anion radical scavenging activity
Superoxide radical (O2-) was generated from the photoreduction of riboflavin and was deducted by nitro blue tetrazolium dye (NBT) reduction method. Measurement of superoxide anion scavenging activity was performed based on the method described by Winterbourne et al 186. The assay mixture contained sample with 0.1ml of Nitro blue tetrazolium (1.5 mM NBT) solution, 0.2 ml of EDTA (0.1M EDTA), 0.05 ml riboflavin (0.12 mM) and 2.55 ml of phosphate buffer (0.067 M phosphate buffer). The control tubes were also set up where in DMSO was added instead of sample. The reaction mixture was illuminated for 30 min and the absorbance at 560 nm was measured against the control samples. Quercetin was used as the reference compound. All the tests were performed in triplicate and the results averaged. The percentage inhibition was calculated by comparing the results of control and test samples.
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Total antioxidant activity (Phosphomolybdic acid method)187
The antioxidant activity of the sample was evaluated by the transformation of Mo (VI) to Mo (V) to form phosphomolybdenum complex. An aliquot of 0.4 ml of sample solution was combined in a vial with 4 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The vials were capped and incubated in a water bath at 950C for 90 min. After the samples had cooled to room temperature, the absorbance of the mixture was measured at 695 nm against a blank. The antioxidant activity was expresses relative to that of ascorbic acid.
Determination of Hydroxyl radical scavenging activity
This was assayed as described by Elizabeth and Rao.188 The assay is based on quantification of degradation product of 2-deoxy ribose by condensation with TBA. Hydroxyl radical was generated by the Fe3+ -Ascorbate -EDTA -H2O2 system (Fenton reaction). The reaction mixture contained 0.1 ml deoxyribose (2.8mM),0.1 ml EDTA (0.1 mM), 0.1 ml H2O2 (1mM), 0.1 ml Ascorbate (0.1mM), 0.1 ml KH2PO4-KOH buffer, pH 7.4 (20mM) and various concentrations of plant extract in a final volume of 1 ml. The reaction mixture was incubated for 1 hour at 370 C. Deoxyribose degradation was measured as TBARS and the percentage inhibition was calculated.
Determination of Nitric oxide radical scavenging activity
Nitric oxide generated from sodium nitroprusside in aqueous solution at physiological pH interacts with oxygen to produce nitrite ions, which were measured by the method of Garrat.189 The reaction mixture (3ml) containing 2 ml of sodium nitroprusside (10mM), 0.5 ml of phosphate buffer saline (1M) were incubated at 250C for 150 mins. After incubation, 0.5 ml of the reaction mixture containing nitrite was pipetted and mixed with 1 ml of sulphanilic acid reagent (0.33%) and allowed to stand for 5 min for completing diazotization. Then 1 ml of naphthylethylene diamine dihydrochloride (1% NEDA) was added, mixed and allowed to stand for 30 mins. Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide, which interacts with oxygen to produce nitrite ions which can be estimated by the use of Griess Illosvery reaction at 540 nm.
A modified method of Benzie and Strain 190 was adopted for the FRAP assay. The stock solutions included 300 mM acetate buffer, pH 3.6, 10 mM TPTZ (2, 4, 6-tripyridyl-S-triazine) solution in 40 mMHCl and 20 mMFecl3. 6H2O. The fresh working solution was prepared by mixing 25 ml acetate buffer, 2.5 ml TPTZ and 2.5 ml Fecl3 .6H2O. The temperature of the solution was raised to 370 C before using. Plant extracts (0.15 ml) were allowed to react with 2.85 ml of FRAP solution for 30 min in the dark condition. Readings of the colored product (Ferrous tripyridyltriazine complex) were taken at 593 nm. The standard curve was linear between 200 and 1000 µM Feso4. Results are expressed in µM (Fe (II) /g dry mass and compared with that of ascorbic acid.
Iron chelating activity
The method of Benzie and strain190 was adopted for the assay. The principle is based on the formation of O-Phenanthroline-Fe2+ complex and its disruption in the presence of chelating agents. The reaction mixture containing 1 ml of 0.05% O-Phenanthroline in methanol, 2 ml ferric chloride (200µM) and 2 ml of various concentrations ranging from 10 to 1000µg was incubated at room temperature for 10 min and the absorbance of the same was measured at 510 nm. EDTA was used as a classical metal chelator. The experiment was performed in triplicates.
Estimation of total phenol
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The measurement of total phenol is based on Mallick and Singh.191 To 0.25g of sample, added 2.5 ml of ethanol and centrifuged at 2oC for 10 mins. The supernatant was preserved. Then, the sample was re-extracted with 2.5 ml of 80% ethanol and centrifuged. The pooled supernatant was evaporated to dryness. Then, added 3 ml of water to the dried supernatant. To which added 0.5 ml of Folins phenol reagent and 2 ml of sodium carbonate (20%). The reaction mixture was kept in boiling water bath for 1 min. the absorbance was measured at 650 nm in a spectrophotometer.
Estimation of total flavonoids 192
0.2g of the plant material was ground with ethanol-water in 2 different ratios namely 9:1 and 1:1 respectively. The homogenate was filtered and these 2 ratios were combined. This was evaporated to dryness until most of the ethanol has removed. The resultant aqueous extract was extracted in a separating funnel with hexane or chloroform. The solvent extracted aqueous layer was concentrated 0.5 ml of aliquot of extract was pipette-out in a test tube. 4 ml of the vanillin reagent (1% vanillin in 70% conc. H2SO4) was added and kept in a boiling water bath for 15 mins. The absorbance was read at 360 nm. A standard was run by using catechol (110 µg/ml).