Procedure.
Each pair of students will assay one flavonoid using the methodology described
below for its antioxidant activity and compare the obtained results with those on
Table 1
0.0208g
DPPH Photometric Assay.
Sample stock solution (1.0 mg/mL) has to be diluted to final concentrations of 100,
50, 10, 5, 1 and 0.5 pg/mL, in ethanol. One mL of a 0.5 mM DPPH ethanol solution
has to be added to 2.5 mL of sample solutions of different concentrations, and
allowed to react at room temperature. After 30 min the absorbance values are to be
measured at 518 nm and converted into the percentage antioxidant activity (AA)
using the following formula:
C
AA% = 100-{[(Abs sample – Abs blank) X100]/Abs control
Ethanol (1.0 mL) plus flavonoid solution (2.5 mL) is used as a blank. DPPH solution
(1.0 mL; 0.5 mm) plus ethanol (2.5 mL) is used as a negative control. The EC50
values must be calculated by linear regression of plots where the abscissa
represented the concentration of tested plant extracts and the ordinate the average
percent of antioxidant activity from three separate tests (Mensor et al., 2001).
Statistical analysis. The experiments are to be done in triplicate. The results should
be given as mean standard deviation (SD). Student’s t-test mu be used for
comparison between two means and a one-way analysis of variance (ANOVA) must
be used for comparison of more than two means (Runyon and Haber, 1984). A
difference is considered statistically significant when p s 0.05.
Neg
Control val
こ
=
1.584
p Type her
DPPH
DPPH
0.5 mm
{
[
with DPPH, after 30 minutes of incubation.
Table 1: Values obtained for the solutions of pre-tested flavonoids in reaction
0.5 DPPH
DPPH DPPH
mM 0.5 mm
0.5
0.5 DPPH
100 50
mM
10
mm 0.5 mm
ug/L
5
pg/mL
1
ug/mL
0.5
pg/mL
pg/mL
ug/mL
0.957 1.048 1.140
0.961
1.150
1.044
1.159 1.250
1.135 1.150
0.954
1.161
1.051
1.249
1.146
1.151
0.001
1.159 1.248
1.281
0.868 0.874 0.899
0.955
0.986 1.015
0.867 0.877 0.895 0.961 0.985 1.018
0.868 0.872 0.899 0.954 0.987 1.016
0.002
1.167
0.181 0.227 0.259 0.635 0.883
0.945
0.179 0.227 0.258 0.633 0.881 0.945
0.180 0.225 0.257 0.632 0.878 0.943
0.003
1.092
0.296
0.269
0.328 0.467 0.644 0.905
0.269 0.293 0.329 0.463 0.644 0.901
0.294 0.329 0.464
0.268
0.642 0.901
0.004
1.085
0.388
7.14 1.684
0.055 0.07
2.oll
10.07 0.982 0.0670 1.436 1.768 1.899
10.078 0.141 0.081877 1.314 1H43 1.543
Concentration
Naringenin
Naringenin
Naringenin
Blank
Negative
Apigenin
Apigenin
Apigenin
Blank
Negative
Kaempferol
Kaempferol
Kaempferol
Blank
Negative
Luteolin
Luteolin
Luteolin
Blank
Negative
Quercetin
Quercetin
Quercetin
Blank
Negative 11.584
Catechin
0.202 0.223
Catechin
0.201 0.224
Catechin
0.203 0.224
Flank
0.008
egative
1.154
[.
0.303
0.305
0.305
0.485
0.483
0.483
0.986
0.984
0.983
1.059
1.050
1.049
1
1.015
0.992
0.9373333
0.805
1.025
42.1147%
39.5806%
0.5
1059
0944
1976
For each Flavonoid, please calculate:
A4% = 100 – Average Abs sample – Abs blanklx 100)
Abs Neg Control
100
AAS
Y = 2.477in(x) • 15,416
R? 09388
100
05
1 5 10
50
Concentration
And Plot the graphic for all flavonoids in order to answer the Post-Practical Test
-OENITI
ALL
SAFETY NOTES: Please read before commencing work!
Avoid breathing vapours.
Keep flammable materials away from heat/sparks/open flames. Wash skin
thoroughly after handling any chemicals
Always use appropriate PPE
Ethanol
Flammable liquid and moderately toxic by
ingestion. Keep away from all ignition sources.
{
DPPH solution –
May cause sensitization by inhalation and by skin
contact. As it is harmful, do not breathe the DPPH powder. Do not empty into drains.
[
a
Quercetin –
Toxic if swallowed.
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