Analytical Estimation

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DPPH RADICAL SCAVENGING ACTIVITY (Shimada et al.

, 1992)
Principle
DPPH radical is scavenged by antioxidants through the donation of a proton
forming the reduced DPPH. The colour change from purple to yellow after reduction
can be quantified by its decrease in absorbance at wavelength 517 nm.
Reagents
1. 0.2 mM DPPH
2. 80% Methanol
3. Butylated Hydroxy Anisole
Procedure
Various concentrations of ethanol & aqueous extract of the sample (4.0 ml)
were mixed with 1.0 ml of methanolic solution containing DPPH radicals, resulting in
the final concentration of DPPH being 0.2 mM. The mixture were shaken vigorously
and left to stand for 30 min, and the absorbance was measured at 517 nm. BHA was
used as control. The percentage of DPPH decolorization of the sample was calculated
according to the equation:
% decolorization = [1-(ABS sample /ABS control)] x 100
IC 50 value (mg extract/ml) is the inhibitory concentration at which DPPH
radicals were scavenged by 50 %. Ascorbic acid and BHA were used for comparison.

ABTS RADICAL SCAVENGING ACTIVITY (Re et al., 1999)


Principle
ABTS decolourisation assay involves the generation of the AABTS +,
chromophore by the oxidation of ABTS with ammonium persulphate. It is applicable
for both hydrophilic and lipophilic compounds. The scavenging activity of the plant
extracts on ABTS radical action were measured at 734 nm.
Reagents
1. 7mM ABTS
2. 2.45 mM Ammonium per sulphate
3. ABTS solution: 7mM of ABTS was mixed with 2.45 mM ammonium per
sulphate and the mixture were allowed to stand in dark at room temperature for
12-16 hours before use. ABTS+ solution were diluted to an absorbance of
0.7±0.05 with ethanol at 734 nm.
4. Ethanol
Procedure
Samples were diluted to produce 0.2 to 1.0 mg/ml. The reaction was initiated by the
addition of 1.0 ml of diluted ABTS- to 10 µl of different concentration of ethanolic and
aqueous extract of the sample or 10 µl of methanol as control. The absorbance was read
at 734 nm and the percentage inhibition was calculated. The inhibition was calculated
according to the equation I = A1/A0 x 100, where A0 is the absorbance of control
reaction, A1 is the absorbance of test compound.

SUPEROXIDE RADICAL SCAVENGING ACTIVITY (Liu t al., 1997)


The superoxide radical scavenging activity was analysed by the method of Liu
et al., 1997.
Principle
Superoxide radical was generated from the photo reduction of riboflavin and
was detected by NBT reduction method.
Reagents
1. 6µm EDTA
2. 3 µg NACN
3. 2 µM riboflavin
4. 2 µM NBT
5. 67 µM KH2PO4-Na2HPO4 buffer, pH7.8.
Procedure
The reaction mixture contained 6µm EDTA, 3 µg NACN, 2 µM riboflavin, 2
µM NBT, 67 µM KH2PO4 -Na2HPO4 buffer, pH7.8 and various concentration of the
extracts in a final volume of 3.0 ml. The tubes were illuminated under incandescent
lamp for 15 mins. The optical density at 560nm was measured before and after
illumination. The inhibition of superoxide radical was determined by comparing the
absorbance values of the control with those of the treatments. ascorbic acid was used as
standard.
APPENDIX-25
NITRIC OXIDE RADICAL SCAVENGING ASSAY ( Madan et al (2005))
Principle
The interaction of ethanolic extract of the sample with nitric oxide was
assessed by the nitrite detection method. Nitric oxide was generated from sodium nitro
prusside and measured by Griess illosvory reaction. Sodium nitroprusside in aqueous
solution at physiological pH spontaneously generated nitric oxide, which interacts with
oxygen to produce nitrite, which can be estimated by the use of Griess illosvory
reagent. In the present experiment, nitrite ion was measured by using Griess illosvory
reagent, which is modified by using naphthyl ethylene diamine dihydro chloride instead
of 1-naphthyl amine.
Reagents
1. Sodium nitroprusside solution (10mM; 0.2998gm of sodium nitroprusside was
accurately weighed and dissolved in distilled water to make up the volume to
100ml in a volumetric flask.
2. Naphthyl ethylene diamine dihydro chloride (NEDD) (0.1%) weighed
accurately 0.1gm of NEDD and dissolved in 60ml of 50% glacial acetic acid by
heating and made up the volume to 100ml in a volumetric flask with distilled
water.
3. Sulphanilic acid(0.33% w/v) reagent: 0.33g of sulphanilic acid was dissolved in
100mlof 20% glacial acetic acid by heating.
4. Phosphate buffer saline (PBS) pH.7
5. Dimethyl sulfoxide (DMSO), distilled.
Procedure
Nitric oxide generated from sodium nitroprusside in aqueous solution at
physiological pH interacts with oxygen to produce nitrite ions whi ch are measured at
540nm. The reaction mixture (6.0 ml) containing sodium nitroprusside (4.0 ml)
phosphate buffer saline(PBS,1.0ml) and extract (1.0 ml at various concentrations) in
DMSO was incubated at 25 0c for 15 mins. After incubation 0.5 ml of the r eaction
mixture was removed, 1.0 ml of sulphanilic acid reagent was added, mixed well and
allowed to stand for 5 mins for the completion of diazotization. Then add 1.0ml NEDD
and stand for 30 mins in diffused light. A pink coloured chromophore formed was
measured at 540nm against corresponding blank solution. Ascorbic acid was used as
standard.
APPENDIX-26
HYDROGEN PEROXIDE SCAVENGING ACTIVITY
Principle
Hydrogen peroxide H2O2 generated a singlet oxygen (O2) and a hydroxyl radical (OH -)
, which then become powerful oxidising agents, they can cross membranes and may
oxidize a number of compounds , while H2O2 itself cannot react , it can generate the
highly reactive hydroxyl radical (OH), through the fenton reaction . thus the scavenger
of H 2O2 is an important anti –oxidant defense mechanism.
Fe2+ + H2O2 → Fe3+ + OH + OH-
the decomposition of H2O2 to water involves the transfer of electrons.
→2
H2O2 + 2H+ + 2e+ H2O2.
The Scavenging Of H2O2. Was measured at 230 nm in UV/Visible spectrophotometrically.
Reagents required:-
1.Standard solution:-
50mg of Ascorbic acid is dissolved in 50ml standard flask using distilled water.
(conc., 1mg/ml)
2.Extract solution:-
50mg of methanolic dried extract is dissolved in 50ml standard flask using distilled
water. (conc., 1mg/ml)
3. 43mM H2O2. Is prepared with PBS ( pH -7.4)
PROCEDURE:-
1.Prepare (50 - 250µg ) concentration of standard and extract solution. From
that take 3.4 ml of aliquot respectively.
2. Add 0.6 ml of H2O2 .
3. Incubate At Room Temparature For 10 Mins .
4. Read at 230 nm in UV /Visible spectophotometry.
5. 3.4 ml of buffer and 0.6 ml of H2O2 alone serves as blank solution.
APPENDIX-27
HYDROXYL RADICAL SCAVENGING ASSAY (Smirnoff and Cumbes, 1989)
Principle
OH radicals were generated from FeSO4 and hydrogen peroxide and detected by
their ability to hydroxylate salicylate and the hydroxylated salicylate complex was
measured at 562 nm.
Reagents
1. 1.5 mM Ferrous sulphate
2. 6 mM Hydrogen peroxide
3. 20 mM sodium salicylate
Procedure
The reaction mixture 3.0 m contained 1.0 ml of 1.5 mM FeSo 4, 0.7 ml of 6 mM
hydrogen peroxide, 0.3 ml of 20 mM sodium salicylate and varying concentrations of
the extract. After incubation for 1 hour at 37°C, the absence of the hydroxylated
salicylate complex was measured at 562 nm. The percentage scavenging effect was
calculated as
Scavenging activity = [1-(A1 -A2)/A0] x 100%
Where A0 was absorbance of the control (without extract) and A1 was the
absorbance in the presence of the extract, A2 was the absorbance without sodium
salicylate.

principle

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