O. P. Mishra et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5325–5328
5327
radicals, and chelate transition metals, resulting in complexes
which are redox inactive in the Fenton reaction.24 Since such a
compound may have diverse antioxidant properties, we chose to
evaluate our synthetic agents using various assays.25 In light of
these observations, we proceeded to evaluate the reducing power
of synthesized (S,S)-SDG-1 and (R,R)-SDG-2 as well as their free
radical scavenging activity against hydroxyl, peroxyl, and DPPH
free radicals.
substrate concentration was observed at lower concentrations
(1–100 M), allowing regression line equations to be established
for the five compounds. This allowed the half maximal effective
concentration (EC50) for reducing power to be calculated (Fig. 5).
The EC50 (mean std. dev.) values for (S,S)-SDG-1 and (R,R)-SDG-
l
2 were 292.17 27.71
These values were comparable to that of natural (S,S)-SDG-1
(EC50 = 275.24 13.15 M) but approximately threefold higher
than that exhibited by ascorbic acid (EC50 = 1129.32 88.79 M)
and -tocopherol (EC50 = 944.62 148.00 M).
lM and 331.94 21.21 lM, respectively.
l
The reducing power of synthetic (S,S)-SDG-1, synthetic (R,R)-
l
SDG-2, natural (S,S)-SDG-1, ascorbic acid, and
a
-tocopherol was
a
l
determined by the reduction of K3FeCN6 in the presence of FeCl3,
as measured by the absorbance of the resulting ferric-ferrous com-
plex (Fig. 4).26 The reducing power of synthetic (S,S)-SDG-1, syn-
thetic (R,R)-SDG-2, and natural (S,S)-SDG-1 was significantly
concentration-dependent at higher concentrations; however, at
all concentrations tested, the SDGs had comparable or higher
reducing power than known antioxidants ascorbic acid and
The ability of synthetic (S,S)-SDG-1 and (R,R)-SDG-2 to scavenge
hydroxyl and peroxyl radicals as manifested by their inhibition of
the oxidation of fluorescein was assessed by the Hydroxyl Radical
Antioxidant Capacity (HORAC, gallic acid standard) and Oxygen
Radical Antioxidant Capacity assays (ORAC, Trolox standard),
respectively (Table 1). Fluorescein oxidation by hydroxyl radicals
was decreased by synthetic (S,S)-SDG-1 and synthetic (R,R)-SDG-
2 in a concentration-dependent manner and was found to be two-
fold higher than gallic acid. However, synthetic (S,S)-SDG-1 activity
differed from natural (S,S)-SDG-1, likely due to trace impurities.
Fluorescein oxidation by peroxyl radicals generated using 2,20-azo-
bis(2-amidinopropane) dihydrochloride (AAPH) was greatly re-
duced in the presence of synthetic (S,S)-SDG-1, (R,R)-SDG-2 and
natural (S,S)-SDG-1, with a twofold increase in potency over the
Trolox standard (Table 1).
a-tocopherol, with a notable increase in potency in the 200–
500 M range. A linear relationship between reducing power and
l
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00
natural (S,S)-SDG-1
synthetic (R,R)-SDG-2
synthetic (S,S)-SDG-1
ascorbic acid
**
**
The free radical scavenging activities of synthetic (S,S)-SDG-1
and (R,R)-SDG-2 were determined using a 2,2-diphenyl-1-pic-
rylhydrazyl (DPPH) free radical scavenging assay and were
compared to those of natural (S,S)-SDG-1, ascorbic acid, and
α-tocopherol
*
*
a
-tocopherol (Fig. 6). At low (5–25
(50–100 M) ranges, the SDGs exhibited similar scavenging poten-
tials; however, at higher concentrations (250–500 M), the inhibi-
lM) and mid-concentration
*
*
l
*
*
*
*
*
*
l
tion by synthetic (S,S)-SDG-1 was significantly lower than those
exerted by (R,R)-SDG-2 and natural (S,S)-SDG-1. Establishing
regression lines for the potentials at low- and mid-concentration
0
1
25
50
100
250
500
Concentration (µM)
ranges (5–100
of these compounds to be determined. As shown in Figure 7, syn-
thetic (R,R)-SDG-2 (123.63 8.67 M) and synthetic (S,S)-SDG-1
(157.54 21.30 M) were not significantly different. These values
were similar to those exhibited by natural (S,S)-SDG-1 (83.94
2.80 M) and -tocopherol (132.81 12.57 M) but considerably
M). These
lM) allowed the free radical EC50 scavenging activity
Figure 4. Reducing power of synthetic (S,S)-SDG-1 and (R,R)-SDG-2. The increase in
absorbance at 700 nm indicates increase in reducing power. The results are
presented as mean standard deviation (n = 3). ⁄p <0.05 significantly lower than
natural (S,S)-SDG-1, synthetic (R,R)-SDG-2, and synthetic (S,S)-SDG-1; ⁄⁄p <0.05
significantly higher than synthetic (R,R)-SDG-2, synthetic (S,S)-SDG-1, ascorbic acid
and a-tocopherol. The somewhat higher potency of natural (S,S)-SDG-1 may be due
to an unknown impurity in our samples, however the NMR spectra of both natural
and synthetic (S,S)-SDG-1 did not reveal major impurities.
l
l
l
a
l
lower than that shown by ascorbic acid (439.56 11.81
l
results are comparable to those recently reported for SDG.3
In summary, we have synthesized (S,S)-SDG-1 and (R,R)-SDG-2
and characterized their antioxidant properties. Both possess strong
reducing power and high free radical scavenging activity for hydro-
1400
*
1200
*
1000
800
600
400
200
0
Table 1
Antioxidant capacity of synthetic and natural SDGs
Entry
Antioxidant
Against hydroxyla
radicals (GAE)c
Against peroxylb
radicals (TE)d
1
2
3
Natural (S,S)-SDG-1
Synthetic (R,R)-SDG-2
Synthetic (S,S)-SDG-1
3.68 0.27
1.96 0.27
2.09 0.16
2.55 0.11
2.20 0.10
3.03 0.04
Antioxidant capacity of synthetic (S,S)-SDG-1 and (R,R)-SDG-2. Hydroxyl radicals
were generated from hydrogen peroxide by Fenton reaction. Peroxyl radicals were
generated by AAPH (2,20-azobis(2-amidinopropane) dihydrochloride). Oxidation of
fluorescein was measured. Calculations used SDG concentrations that fitted the
linear part of the calibration curve. The results are presented as mean standard
deviation (n = 3). The somewhat higher potency of natural (S,S)-SDG-1 may be due
to an unknown impurity in our samples, however the NMR spectra of both natural
and synthetic (S,S)-SDG-1 did not reveal impurities.
Figure 5. Reducing power of synthetic (S,S)-SDG-1 and (R,R)-SDG-2. The rate of
reaction is linear in the concentration range of 1ꢀ100
lM. Equation of the linear
a
Determined by HORAC assay.
Determined by ORAC assay.
Gallic acid equivalents.
regression was used to determine EC50. The results are presented as mean stan-
dard deviation (n = 3). Natural (S,S)-SDG-1, synthetic (R,R)-SDG-2, and synthetic
(S,S)-SDG-1 were not significantly different from each other. ⁄p <0.05 significantly
higher than natural (S,S)-SDG-1, synthetic (R,R)-SDG-2, and synthetic (S,S)-SDG-1.
b
c
d
Trolox equivalents.