2 October 2023
Antioxidant Results
Methods
DPPH Free-Radical Scavenging Activity Assay
The DPPH radical scavenging activity was measured in a solvent system buffered at pH 6.0 using a 50
mmol/L sodium phosphate buffer (R&M Chemicals, Subang, Malaysia) and ethanol (95%) at a 1:1 (v/v)
ratio (Santos et al., 2017). In a 96-well microplate, 260 µL of DPPH radical (Alfa Aesar, Haverhill, MA,
USA) solution (115 µM) was mixed with 40 µL of compounds disolved in ethanol at various
concentrations (1–1000 µg/mL). The positive control was 0.6–50 µg/mL ascorbic acid (R&M Chemicals,
Subang, Malaysia). After 30 min reaction at room temperature, absorbance was taken at 525 nm
against the blank. The percentage of DPPH scavenging activity was calculated using the following
formula:
𝐷𝑃𝑃𝐻 𝑠𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (%) =
𝑎𝑏𝑠0 − 𝑎𝑏𝑠𝑐𝑜𝑚𝑝𝑜𝑢𝑛𝑑
𝑎𝑏𝑠0
𝑋 100 -
where
abs0 = absorbance of control
abscompound = absorbance of compound
A plot of % DPPH scavenging activity against concentration was constructed to evaluate the activity–
concentration curve (Supplementary; Figure 1). GraphPad Prism (version 9) was used to compute the
concentration of compound required to reduce the DPPH radical by 50% (IC50) .
Table 1: Concentration of compound required to reduce the DPPH radical by 50%. Ascorbic acid was
used as a positive control. n=3 independent experiment
Compounds
Ascorbic acid
Cu2
Ni2
S2
Zn2
Cu4
Ni4
S4
Zn4
IC50 (µg/mL)
2.76±0.17
269.9±48.6
84.8±19.6
18.0±6.2
37.6±4.3
76.5±22.4
63.7±10.0
20.5±2.5
37.7±7.5
Supplementary 1: Plot of % DPPH scavenging activity against concentration of compounds
a) ascorbic acid
100
50
0
1
10
100
-50
c) Ni2
DPPH radical scavenging activity (%)
b) Cu2
100
100
50
50
0
0
1
10
100
1000
10000
-50
1
10
100
1000
10000
10
100
1000
10000
10
100
1000
10000
10
100
1000
10000
-50
d) S2
e) Zn2
100
100
50
50
0
0
1
10
100
1000
10000
-50
1
-50
f) Cu4
g) Ni4
100
100
50
50
0
1
10
100
1000
10000
0
1
ug/mL
-50
-50
h) S4
i) Zn4
100
100
50
50
0
0
1
-50
10
100
1000
10000
1
-50
Concentration (μg/mL)
Reference:
Santos, J.S., Alvarenga Brizola, V.R., Granato, D., 2017. High-throughput assay comparison and
standardization for metal chelating capacity screening: A proposal and application. Food
Chem. 214, 515–522. https://doi.org/10.1016/j.foodchem.2016.07.091