Analysis Of Tris Hcl, L-Ascorbic Acid

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TRIS Hcl, NBT, NADH, Ethanol, PMS, l-ascorbic acid
1. The superoxide radicals is generated in 3 ml of TrisHCl buffer (16 mM, pH 8.0) containing 1 ml of NBT (50 μM), 1 ml NADH (78 μM) and ethanol extract of sample (25 – 50 μg).
2. The reaction was started by adding 1 ml of PMS solution (10 μM) to the mixture.
3. The reaction mixture was incubated at 25° C for 5 min, the absorbance was read at 560 nm using a spectrophotometer (Schimadzu UV-Vis 1700) against blank samples using l- ascorbic acid as a control.
4. Decreased absorbance of the reaction mixture indicated increasing superoxide anion scavenging activity.
5. The percentage inhibition of superoxide anion generation was calculated using the following formula: % inhibition = [(A0-A1)/A0] x 100 where A0 was the absorbance of the control (l- ascorbic acid), and A1 was the absorbance in the presence of ethanol extract of sample or standards.
Dpph assay:
DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical method is an antioxidant assay based on electron-transfer that produces a violet solution in ethanol . This free radical, stable at room temperature, is reduced in the presence of an
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caudatum was measured according to the method proposed by Benzie and Strain. FRAP reagent was prepared by mixing in 25 mL acetate buffer (30 mM; pH 3.6), 2.5 mL TPTZ solution (10 mM) and 2.5 mL ferric chloride solution (20 mM). The mixture was incubated for 15 min at 37 °C before use. Ascorbic acid (vitamin C) was employed as a standard in this assay, and its calibration curve was obtained by using its concentrations ranging from 50 mg/L to 500 mg/L in water. To 2.85 mL FRAP reagent in a test tube, 150 μL plant sample (0.1 mg/mL, in methanol) or standard was added. The mixture was incubated for 30 min in the dark, and its absorbance was measured at 593 nm. The blank contained an equal volume of methanol instead of the plant

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