SANTOSH KUMAR NAGALAPUR.pmd - THE BIOSCAN

SANTOSH KUMAR NAGALAPUR AND S. PARAMJYOTI
gallic acid as the standard phenolic compound.
1 mL of extract solution containing 1g extract in the volumetric
flask was diluted with 46 ml of distilled water. 1mL of Folin-
Ciocalteau reagent was added and the content of flask was
mixed thoroughly. 3 minutes later 3 mL of 2% saturated sodium
carbonate was added to the mixture and was allowed to stand
for 2 hr with intermittent shaking. The absorbance of the blue
color developed was read at 760 nm. The concentration of
the total phenols was expressed as mg/g of dry extract (Kim et
al., 2003). The concentration of total phenolic compounds in
the extract was determined as µg of gallic acid equivalent
using an equation obtained from the standard gallic acid curve.
Absorbance = 0.0008 × gallic acid (µg).
Determination of total flavanoids
1mg samples were added to 1mL of 80% ethanol. Aliquot of
0.5mL was added to the test tubes containing 0.1 mL of 10%
aluminum nitrate, 0.1 mL of 1 M potassium acetate, and 4.3
mL of 80% ethanol. The absorbance of the supernatant was
measured at 415 nm after 40 minutes of incubation at room
temperature. Total flavonoid concentration was calculated
using quercetin as standard (Nieva Moreno et al., 2000).
Absorbance = 0.002180 × µg quercetin – 0.01089.
Ferric reducing activity
Different concentrations of extracts (20µg, 100µg and 200µg)
were mixed with 2.5 mL of 200 mmol/L sodium phosphate
buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide. The
mixture was incubated at 50ºC for 20 minutes then 2.5 mL of
10% trichloroacetic acid (w/v) was added, the mixture was
centrifuged at 1000 rpm for 8 minutes, the upper layer was
mixed with 5 mL of deionised water and 1 mL of 0.1% of ferric
chloride, and the absorbance was measured
spectophotometrically at 700 nm. (Barros et al., 2007). Higher
absorbance of the reaction mixture indicated greater reducing
power.
DPPH radical scavenging activity
Different concentrations of leaf extracts and Butylated hydroxyl
anisole (BHA) (20µg, 100µg and 200µg) were taken and the
volume was adjusted to 100µL by adding methanol. 5 mL of
0.1 mM methanolic solution of 1, 2-Diphenyl picryl hydrazine
(DPPH) was added. The mixture was shaken vigorously and
allowed to stand for 30 minutes in dark. 0.01 mM solution of
DPPH in methanol was used as control, whereas BHA was
used as a standard. The reduction of DPPH radical was
determined by measuring the absorption at 517 nm (Barros et
al., 2007). The radical scavenging activity (RSA) was calculated
as a percentage of DPPH discoloration using the equation:
(Absorbance of control - Absorbance of
sample)
% RSA= X 100.
(Absorbance of control)
IC value for the extract i.e. the concentration at which it
50
scavenges 50 % of DPPH radical was calculated.
Hydroxyl radical scavenging activity
Different concentrations of leaf extracts and BHA (20 µg, 100µg
and 200µg) were taken and the volume was adjusted to 250µL
with 0.1 M phosphate buffer (pH 7.4). 1 mL of iron - EDTA
solution (0.13% ferrous ammonium sulfate and 0.26% EDTA),
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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 solutions. The reaction was initiated by adding 0.5 mL
of 0.22% ascorbic acid. The reaction mixtures were incubated
at room temperature for 30 minutes. The reaction was
terminated by the addition of 1mL of ice-cold TCA (17.5% w/
v). 3mL of Nash reagent was added and the reaction mixture
was allowed to stand at room temperature for 15 minutes for
colour development. The intensity of the yellow colour formed
was measured spectrophotometrically at 412 nm against the
reagent blank (Singh et al., 2002). The percentage of hydroxyl
radical scavenging activity (HRSA) was calculated by using
following equation:
% HRSA=
1- (Absorbance of sample)
(Absorbance of Blank)
X 100.
IC value of the extract was calculated as mentioned earlier.
50
Statistics
The experiments were done in triplicate. Data are expressed
as mean ± SEM. One- way analysis of variance (ANOVA) was
used and the Dunnet’s test was used for comparison of mean
values. All tests were considered to be statistically significant
at p