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Antioxidant Properties of Camphene-Based Thiosemicarbazones: Experimental and Theoretical Evaluation

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The thiosemicarbazone derivatives have a wide range of biological activities, such as antioxidant activity. In this study, the antiradical activities of six camphene-based thiosemicarbazones (TSC-1~6) were investigated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and peroxyl radical scavenging capacity (PSC) assays, respectively, and the results reveal that TSC1~6 exhibited good abilities for scavenging free radicals in a dose-dependent way. Compound TSC-2 exhibited the best effect of scavenging DPPH radical, with the lowest EC50 (0.208 ± 0.004 mol/mol DPPH) as well as the highest bimolecular rate constant Kb (4218 M−1 s−1), which is 1.18-fold higher than that of Trolox. Meanwhile, TSC-2 also obtained the lowest EC50 (1.27 µmol of Trolox equiv/µmol) of scavenging peroxyl radical. Furthermore, the density functional theory (DFT) calculation was carried out to further explain the experimental results by calculating several molecular descriptors associated with radical scavenging activity. These theoretical data suggested that the electron-donating effect of the diethylamino group in TSC-2 leads to the enhancement of the scavenging activities and the studied compounds may prefer to undergo the hydrogen atom transfer process.
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molecules
Article
Antioxidant Properties of Camphene-Based
Thiosemicarbazones: Experimental and
Theoretical Evaluation
Lijuan Yang 1, Haochuang Liu 1, Dasha Xia 2and Shifa Wang 1, 3, *
1College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China;
lijuan_yang@163.com (L.Y.); lhchuang96@163.com (H.L.)
2Hangzhou Yanqu Information Technology Co., Ltd., Hangzhou 310012, China; xiadasha@hotmail.com
3Co-Innovation Center of Ecient Processing and Utilization of Forest Resources, College of Chemical
Engineering, Nanjing Forestry University, Nanjing 210037, China
*Correspondence: wangshifa65@163.com; Tel.: +86-25-8542-8369
Received: 30 December 2019; Accepted: 24 February 2020; Published: 6 March 2020


Abstract:
The thiosemicarbazone derivatives have a wide range of biological activities, such as
antioxidant activity. In this study, the antiradical activities of six camphene-based thiosemicarbazones
(
TSC-1~6
) were investigated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and peroxyl radical scavenging
capacity (PSC) assays, respectively, and the results reveal that
TSC1~6
exhibited good abilities for
scavenging free radicals in a dose-dependent way. Compound
TSC-2
exhibited the best eect
of scavenging DPPH radical, with the lowest EC
50
(0.208
±
0.004 mol/mol DPPH) as well as
the highest bimolecular rate constant K
b
(4218 M
1
s
1
), which is 1.18-fold higher than that of Trolox.
Meanwhile,
TSC-2
also obtained the lowest EC
50
(1.27
µ
mol of Trolox equiv/
µ
mol) of scavenging
peroxyl radical. Furthermore, the density functional theory (DFT) calculation was carried out to
further explain the experimental results by calculating several molecular descriptors associated with
radical scavenging activity. These theoretical data suggested that the electron-donating eect of
the diethylamino group in
TSC-2
leads to the enhancement of the scavenging activities and the studied
compounds may prefer to undergo the hydrogen atom transfer process.
Keywords: camphene-based thiosemicarbazones; DPPH; peroxyl radical scavenging capacity; DFT
1. Introduction
Oxidation is an essential part of aerobic metabolism in living organisms. In the complicated
oxidation process, free radicals are constantly generated containing reactive oxygen species (ROS) and
reactive nitrogen species (RNS) [
1
,
2
]. It is widely known that free radicals play a dual role
in vivo
as both
beneficial and deleterious compounds [
3
5
]. The beneficial eects of free radicals involved important
physiological roles in the function of a number of intercellular signaling pathways etc. [
5
,
6
]. In contrast,
excessive free radicals can cause irreversible tissue injury by attacking biomolecules such as lipids,
proteins, and DNA [
7
,
8
]. Therefore, this balance is essential for the survival of organisms and their
health [
3
]. In order to maintain the balance of the activity of the free radicals in living cells, the organism
has evolved antioxidant defense systems, protecting against free radical damage [
9
,
10
]. However,
when our endogenous defense system has incomplete eciency, or a rise in free radicals under special
conditions (smoking, chemical air pollutants, radiation, and inflammation), the imbalance between
free radicals and antioxidants results in oxidative stress (oxidative damage), which is now believed to
be one of the important factors in the occurrence of certain human diseases including atherosclerosis,
rheumatoid arthritis, cancer, and neurodegenerative diseases [
11
,
12
]. So, antioxidants have aroused
Molecules 2020,25, 1192; doi:10.3390/molecules25051192 www.mdpi.com/journal/molecules
Molecules 2020,25, 1192 2 of 16
the interest of biomedical scientists and clinicians because they protect the body against damage by
harmful free radicals [13]. In addition, it is necessary to explore some eective radical scavengers.
Thiosemicarbazone is a well-known class of ligand with a broad spectrum of biological activities,
including antibacterial, antifungal, antiviral, antimalarial, anticancer, and antioxidant activities [
14
21
].
Therefore, thiosemicarbazone has attracted increasing research attention in the pharmaceutical industry.
As a bicyclic monoterpene, camphene has been broadly employed as a starting material in the synthesis
of biomolecules for its low cost and broad availability [
22
]. Moreover, camphene was reported as
having high hypolipidemic action [
23
] and antioxidant activity [
22
]. In the earlier reports, the synthesis
and potential activity of camphene-based thiosemicarbazone derivatives have been reported [
24
,
25
].
For example, Mariana reported camphene-based thiosemicarbazone compounds with anti-tuberculosis
activity [
26
]. A series of camphene-based thiosemicarbazone derivatives were synthesized and their
antitumor activity was evaluated by our research group [
27
]. Nevertheless, there are few reports on
the antioxidant activities of camphene-based thiosemicarbazones.
Considering the above-mentioned reasons and as an extension of our previous work, in this
work we attempted to experimentally and theoretically evaluate the antioxidant behavior of six
camphene-based thiosemicarbazone compounds (
TSC-1~6
), which were selected from a series
of previously synthesized compounds [
27
]. The experimental results show that the compound
2-hydroxy-4-(N, N-diethylamino) benzaldehyde-4-(2
0
-isocamphanyl) thiosemicarbazone
(TSC-2)
possesses high scavenging radical activity. Furthermore, the DFT approach was employed to elucidate
scavenging free radical mechanisms theoretically [
28
30
]. This study will assist in the design of
thiosemicarbazone derivatives with the desired antioxidant properties.
2. Results and Discussion
The six camphene-based thiosemicarbazone (TSC1-6) are depicted in Figure 1.
Molecules 2020, 25, x 2 of 15
certain human diseases including atherosclerosis, rheumatoid arthritis, cancer, and
neurodegenerative diseases [11,12]. So, antioxidants have aroused the interest of biomedical scientists
and clinicians because they protect the body against damage by harmful free radicals [13]. In
addition, it is necessary to explore some effective radical scavengers.
Thiosemicarbazone is a well-known class of ligand with a broad spectrum of biological activities,
including antibacterial, antifungal, antiviral, antimalarial, anticancer, and antioxidant activities [14–
21]. Therefore, thiosemicarbazone has attracted increasing research attention in the pharmaceutical
industry. As a bicyclic monoterpene, camphene has been broadly employed as a starting material in
the synthesis of biomolecules for its low cost and broad availability [22]. Moreover, camphene was
reported as having high hypolipidemic action [23] and antioxidant activity [22]. In the earlier reports,
the synthesis and potential activity of camphene-based thiosemicarbazone derivatives have been
reported [24,25]. For example, Mariana reported camphene-based thiosemicarbazone compounds
with anti-tuberculosis activity [26]. A series of camphene-based thiosemicarbazone derivatives were
synthesized and their antitumor activity was evaluated by our research group [27]. Nevertheless,
there are few reports on the antioxidant activities of camphene-based thiosemicarbazones.
Considering the above-mentioned reasons and as an extension of our previous work, in this
work we attempted to experimentally and theoretically evaluate the antioxidant behavior of six
camphene-based thiosemicarbazone compounds (TSC-1~6), which were selected from a series of
previously synthesized compounds [27]. The experimental results show that the compound 2-
hydroxy-4-(N, N-diethylamino) benzaldehyde-4-(2-isocamphanyl) thiosemicarbazone (TSC-2)
possesses high scavenging radical activity. Furthermore, the DFT approach was employed to
elucidate scavenging free radical mechanisms theoretically [28–30]. This study will assist in the
design of thiosemicarbazone derivatives with the desired antioxidant properties.
2. Results and Discussion
The six camphene-based thiosemicarbazone (TSC1-6) are depicted in Figure 1.
Figure 1. Molecular structures of the studied compounds TSC-1~6.
2.1. Radical Scavenging Activity
2.1.1. 2,2-diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging Assay
DPPH radical is a stable radical, and has an odd electron with a strong absorption band at 517
nm in ethanol solution. When the DPPH radical is reduced by acquiring an electron or hydrogen
atom from the antioxidant, the color of DPPH alcohol solution turns from deep violet to pale yellow;
meanwhile, the intensity of the absorption peak at 517 nm decreases [31]. As shown in Figure 2a, all
of the compounds displayed appreciable DPPH radical scavenging activity, compared with Trolox.
In the range of 10–100 µM, with the increasing concentration, the DPPH scavenging rate of TSC-1,
TSC-4, TSC-5, and TSC-6 showed considerable dose-dependent activity and reached the maximum
of about 83% at 100 µM. For TSC-2 and TSC-3, they showed higher scavenging activity at 25 µM,
Figure 1. Molecular structures of the studied compounds TSC-1~6.
2.1. Radical Scavenging Activity
2.1.1. 2,2-diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging Assay
DPPH radical is a stable radical, and has an odd electron with a strong absorption band at
517 nm in ethanol solution. When the DPPH radical is reduced by acquiring an electron or hydrogen
atom from the antioxidant, the color of DPPH alcohol solution turns from deep violet to pale yellow;
meanwhile, the intensity of the absorption peak at 517 nm decreases [31]. As shown in Figure 2A, all
of the compounds displayed appreciable DPPH radical scavenging activity, compared with Trolox. In
the range of 10–100
µ
M, with the increasing concentration, the DPPH scavenging rate of
TSC-1
,
TSC-4
,
TSC-5
, and
TSC-6
showed considerable dose-dependent activity and reached the maximum of about
83% at 100
µ
M. For
TSC-2
and
TSC-3
, they showed higher scavenging activity at 25
µ
M, while with
a further increase in concentration the scavenging rate did not show a significant change. Figure 2B
Molecules 2020,25, 1192 3 of 16
shows that in the range of 4–25
µ
M,
TSC-2
and
TSC-3
showed dose-dependent activity. Among these
compounds, TSC-2 and TSC-3 had more remarkable scavenging DPPH activity than others.
Molecules 2020, 25, x 3 of 15
while with a further increase in concentration the scavenging rate did not show a significant change.
Figure 2b shows that in the range of 4–25 µM, TSC-2 and TSC-3 showed dose-dependent activity.
Among these compounds, TSC-2 and TSC-3 had more remarkable scavenging DPPH activity than
others.
Figure 2. (A) 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity (%) of the camphene-
based thiosemicarbazone compounds (TSC-1~6) at different concentrations (10~100 µM); (B) DPPH
radical scavenging capacity (%) of compounds TSC-2, TSC-3, and Trolox at different concentrations
(4~100 µM).
The non-linear regression model [32] was applied to fit the experimental data to obtain the EC50
(Table 1). As showed in Table 1, TSC-2 exhibited the highest DPPH scavenging activity, which is
higher than that of Trolox. The EC50 value of TSC-3 was almost the same compared with Trolox. The
EC50 values of TSC-1, TSC-4, TSC-5, TSC-6 were higher than the reference.
TSC-1 TSC-2 TSC-3 TSC-4 TSC-5 TSC-6 Trolox
0
20
40
60
80
100
Radical Scavenging(%)
10 µM
20 µM
25 µM
30 µM
40 µM
50 µM
60 µM
70 µM
100 µM
(A)
TSC-2 TSC-3 Trolox
0
20
40
60
80
100
Radical Scavenging(%)
4 µM
8 µM
12 µM
16 µM
20 µM
25 µM
30 µM
40 µM
100 µM
(B)
Figure 2.
(
A
) 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity (%) of
the camphene-based thiosemicarbazone compounds (
TSC-1~6
) at dierent concentrations (10~100
µ
M);
(
B
) DPPH radical scavenging capacity (%) of compounds
TSC-2
,
TSC-3
, and Trolox at dierent
concentrations (4~100 µM).
The non-linear regression model [
32
] was applied to fit the experimental data to obtain the EC
50
(Table 1). As showed in Table 1,
TSC-2
exhibited the highest DPPH scavenging activity, which is higher
than that of Trolox. The EC
50
value of
TSC-3
was almost the same compared with Trolox. The EC
50
values of TSC-1,TSC-4,TSC-5,TSC-6 were higher than the reference.
Molecules 2020,25, 1192 4 of 16
Table 1. Scavenging radical activities of compounds TSC-1~6.
Compounds
DPPH Assay PSC Assay
EC50(µM) at
Fixed Time 1
EC50 mol/mol
DPPH Kinetic
Stoichiometric
Factor (n) 2
Bimolecular Rate
Constant 3kb
(M1s1)
EC50 (µM)
PSC Value
(µmol of
Trolox
equiv/µmol)
TSC-1 0.411 0.308 ±0.003 1.29 ±0.23 16.16 ±1.15 62.26 ±1.65 0.34
TSC-2 0.208 0.208 ±0.004 2.27 ±0.13 4218.08 ±551.26 16.78 ±0.12 1.27
TSC-3 0.246 0.226 ±0.002 2.27 ±0.20 308.71 ±19.91 37.78 ±0.13 0.56
TSC-4 0.475 0.398 ±0.006 1.21 ±0.16 16.61 ±0.75 34.62 ±0.58 0.62
TSC-5 0.393 0.341 ±0.015 1.33 ±0.23 22.87 ±2.32 43.33 ±0.99 0.49
TSC-6 0.297 0.302 ±0.015 1.44 ±0.30 45.61 ±4.13 48.38 ±0.12 0.44
Trolox 0.241 0.249 ±0.002 1.86 ±0.12 3527.11 ±103.95 21.38 ±0.25 1
1
The fixed time was one hour;
2
Stoichiometric factor expresses the number of radical scavenged by one molecule
of antioxidant;
3
The bimolecular rate constant (k
b
) was determined by the slope of a linear plot of
(dA/dt)0
against
A0[AH].
The reaction of DPPH radical with antioxidants is basically a kinetic driving process [
33
]. So, it
is necessary to assess the kinetic behavior between DPPH radical and antioxidants. Two processes
occurred, containing a rapid process in the first few seconds followed by a slower one at longer
reaction times. According to the literature [
34
], it was detected that the reaction was best modeled by
an empirical bi-exponential decay function. (Figure 3and Figure S1).
Molecules 2020, 25, x 4 of 15
Table 1. Scavenging radical activities of compounds TSC-1~6.
Comp
ounds
DPPH assay PSC assay
EC50(µM)
at Fixed
Time 1
EC50 mol/mol
DPPH Kinetic
Stoichiometr
ic Factor (n)
2
Bimolecular
Rate Constant 3
kb (M-1 s-1)
EC50 (µM)
PSC Value
(µmol of
Trolox
equiv/µmol)
TSC-1 0.411 0.308 ± 0.003 1.29 ± 0.23 16.16 ± 1.15 62.26 ± 1.65 0.34
TSC-2 0.208 0.208 ± 0.004 2.27 ± 0.13 4218.08 ± 551.26 16.78 ± 0.12 1.27
TSC-3 0.246 0.226 ± 0.002 2.27 ± 0.20 308.71 ± 19.91 37.78 ± 0.13 0.56
TSC-4 0.475 0.398 ± 0.006 1.21 ± 0.16 16.61 ± 0.75 34.62 ± 0.58 0.62
TSC-5 0.393 0.341 ± 0.015 1.33 ± 0.23 22.87 ± 2.32 43.33 ± 0.99 0.49
TSC-6 0.297 0.302 ± 0.015 1.44 ± 0.30 45.61 ± 4.13 48.38 ± 0.12 0.44
Trolox 0.241 0.249 ± 0.002 1.86 ± 0.12 3527.11 ± 103.95 21.38 ± 0.25 1
1The fixed time was one hour; 2 Stoichiometric factor expresses the number of radical scavenged by
one molecule of antioxidant; 3 The bimolecular rate constant (kb) was determined by the slope of a
linear plot of (𝑑𝐴 𝑑𝑡)
against A0[AH].
The reaction of DPPH radical with antioxidants is basically a kinetic driving process [33]. So, it
is necessary to assess the kinetic behavior between DPPH radical and antioxidants. Two processes
occurred, containing a rapid process in the first few seconds followed by a slower one at longer
reaction times. According to the literature [34], it was detected that the reaction was best modeled by
an empirical bi-exponential decay function. (Figure 3 and Figure S1).
0 1200 2400 3600 4800 6000 7200
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
4 µM
8 µM
12 µM
16 µM
20 µM
25 µM
Absorbance (517)
Time (s)
(A)
0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45
0
10
20
30
40
50
60
70
80
90
100
%DPPH radical remaining
mol TSC-2 / mol DPPH
(B)
0 1200 2400 3600 4800 6000 7200
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Absorbance (517)
Time (s)
4 µM
8 µM
12 µM
16 µM
20 µM
25 µM
(C)
0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40
20
30
40
50
60
70
80
90
100
% DPPH radical remaining
mol TSC-3 /mol DPPH
(D)
Figure 3. Reaction kinetics of DPPH radical with TSC-2 (A) and TSC-3 (C); %DPPH radical remaining
at infinite time at the different concentration of TSC-2 (B)and TSC-3 (D).
By analyzing the data as described in the experimental portion, the EC50 (mol AH/mol DPPH),
stoichiometric factor(n) and the bimolecular rate constants(kb) were procured (Table 1), which are
Figure 3.
Reaction kinetics of DPPH radical with
TSC-2
(
A
) and
TSC-3
(
C
); %DPPH radical remaining
at infinite time at the dierent concentration of TSC-2 (B)and TSC-3 (D).
By analyzing the data as described in the experimental portion, the EC
50
(mol AH/mol DPPH),
stoichiometric factor(n) and the bimolecular rate constants(k
b
) were procured (Table 1), which are
Molecules 2020,25, 1192 5 of 16
important parameters to consider when investigating antioxidants. Trolox was used as a reference in
the DPPH kinetic assay. Regarding stoichiometric factor (n), the four tested compounds
TSC-1
,
TSC-4
,
TSC-5
, and
TSC-6
have stoichiometric factors ranging from 1.21 to 1.44. The Trolox has a stoichiometric
factor of 1.86.
TSC-2
and
TSC-3
had higher stoichiometric factor values of almost the same value—2.27.
As illustrated in Figure 4, the
(dA/dt)0
increased linearly with A
0
[AH]. From the slope of a linear plot
of
(dA/dt)0
against A
0
[AH], the bimolecular rate constants (k
b
) for the radical scavenging reaction
were determined and presented in Table 1.
Molecules 2020, 25, x 5 of 15
important parameters to consider when investigating antioxidants. Trolox was used as a reference in
the DPPH kinetic assay. Regarding stoichiometric factor (n), the four tested compounds TSC-1, TSC-
4, TSC-5, and TSC-6 have stoichiometric factors ranging from 1.21 to 1.44. The Trolox has a
stoichiometric factor of 1.86. TSC-2 and TSC-3 had higher stoichiometric factor values of almost the
same value—2.27. As illustrated in Figure 4, the (𝑑𝐴 𝑑𝑡)
increased linearly with A0[AH]. From the
slope of a linear plot of (𝑑𝐴 𝑑𝑡)
against A0[AH], the bimolecular rate constants (kb) for the radical
scavenging reaction were determined and presented in Table 1.
0.0 5.0x10
-6
1.0x10
-5
1.5x10
-5
2.0x10
-5
2.5x10
-5
3.0x10
-5
0.00
0.02
0.04
0.06
0.08
(dA/dt)
0
/ S
-1
A
0
[AH]/ M
TSC-2
Trolox
TSC-3
TSC-6
TSC-5
TSC-1
TSC-4
Figure 4. Linear plots of (dA/dt)0 vs A0[AH] of compounds. A0 is the absorbance at time=0, and [AH]
is the concentration of antioxidant at time=0, R2 > 0.9500.
There were obvious differences in reactivity between the tested compounds. In the combined
reaction kinetics (Figure 3 and Figure S1), it can be found that TSC-2 reacted very quickly with DPPH
with the largest bimolecular rate constant Kb value (4218 M-1S-1), larger than the kb of Trolox (3570 M-
1S-1). The Kb value of TSC-2 is 1.18-fold higher than Trolox, while the Kb value of TSC-3 is one
magnitude less than Trolox. Based on kb value, the sort of scavenging reactivity was TSC-
2>Trolox>TSC-3>>TSC-6>TSC-5 >TSC-1>TSC-4. The results suggest that compounds with higher
stoichiometric factor values tend to have higher Kb values.
Combined with the above results, it could be found that the substituents on the phenyl ring have
a significant effect on the scavenging activity. As observed, the electron-donating groups
(diethylamino group, methoxy group) can dramatically enhanced radical-scavenging activity, which
explains why TSC-2 possessed the highest activity with the most electron-donating diethylamino
group attached on the phenyl ring.
2.1.2. Peroxyl Radical Scavenging Capacity Assay
Peroxyl radical scavenging capacity assay is a simple, rapid and sensitive assay for evaluating
the antioxidant activity of both hydrophilic and lipophilic antioxidants. The reaction mechanism of
this essay has been described in the previous literature [35]. Briefly, the thermal degradation of 2,2-
azobis(2-amidinopropane) dihydrochloride (AAPH) produces peroxyl radicals (ROO•·) which
oxidize non-fluorescent dichlorofluorescein (DCFH) to fluorescent dichlorofluorescein (DCF). The
degree of inhibition of DCFH oxidation by antioxidants was used for evaluating the antioxidant
activity [36].
The peroxyl radical scavenging capacity assays of six tested compounds and Trolox are
determined in Figure 5 and Figure S2.
Figure 4.
Linear plots of (dA/dt)
0
vs. A
0
[AH] of compounds. A
0
is the absorbance at time =0, and
[AH] is the concentration of antioxidant at time =0, R2>0.9500.
There were obvious dierences in reactivity between the tested compounds. In the combined
reaction kinetics (Figure 3and Figure S1), it can be found that
TSC-2
reacted very quickly with
DPPH with the largest bimolecular rate constant K
b
value (4218 M
1
S
1
), larger than the k
b
of Trolox
(3570 M
1
S
1
). The K
b
value of
TSC-2
is 1.18-fold higher than Trolox, while the K
b
value of
TSC-3
is one magnitude less than Trolox. Based on k
b
value, the sort of scavenging reactivity was
TSC-2
>Trolox >
TSC-3
>>
TSC-6
>
TSC-5
>
TSC-1
>
TSC-4
. The results suggest that compounds with
higher stoichiometric factor values tend to have higher Kbvalues.
Combined with the above results, it could be found that the substituents on the phenyl ring have
a significant eect on the scavenging activity. As observed, the electron-donating groups (diethylamino
group, methoxy group) can dramatically enhanced radical-scavenging activity, which explains why
TSC-2
possessed the highest activity with the most electron-donating diethylamino group attached on
the phenyl ring.
2.1.2. Peroxyl Radical Scavenging Capacity Assay
Peroxyl radical scavenging capacity assay is a simple, rapid and sensitive assay for evaluating
the antioxidant activity of both hydrophilic and lipophilic antioxidants. The reaction mechanism
of this essay has been described in the previous literature [
35
]. Briefly, the thermal degradation
of 2,2
0
-azobis(2-amidinopropane) dihydrochloride (AAPH) produces peroxyl radicals (ROO
•·
)
which oxidize non-fluorescent dichlorofluorescein (DCFH) to fluorescent dichlorofluorescein (DCF).
The degree of inhibition of DCFH oxidation by antioxidants was used for evaluating the antioxidant
activity [36].
Molecules 2020,25, 1192 6 of 16
The peroxyl radical scavenging capacity assays of six tested compounds and Trolox are determined
in Figure 5and Figure S2.
Molecules 2020, 25, x 6 of 15
0 5 10 15 20 25 30 35 40
0
500
1000
1500
2000
2500
3000
3500
4000
4500
0255075100
0.2
0.4
0.6
0.8
PSC Unit
Concentration (µM)
Fluorescence
Reaction Time (min)
10 µM
20 µM
30 µM
40 µM
50 µM
100 µM
control
(A)
0 5 10 15 20 25 30 35 40
0
1000
2000
3000
4000
5000
0 50 100 150 200
0.2
0.4
0.6
0.8
1.0
PSC Unit
Concentration (µM)
Fluorescence
Reaction Time (min)
40 µM
50 µM
80 µM
100 µM
200 µM
control
(B)
Figure 5. Time kinetics plots of TSC-2 (A) and TSC-3 (B) inhibition of non-fluorescent
dichlorofluorescein (DCFH) oxidation by 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH).
(the inset is dose-response plots).
It can be seen that ROO oxidizes DCFH to fluorescent DCF over time and antioxidants
scavenged peroxyl radicals and inhibited the oxidation reaction in a dose-dependent way.
Scavenging peroxyl radical activity was expressed as EC50 and as micromoles of Trolox equivalents
per micromole of the test sample. The degree of inhibition was different for the investigated
compounds, as shown in Table 1. Among the six compounds, TSC-2 had the highest PSC value and
that indicated that TSC-2 possess the highest peroxyl radical-scavenging activity, which was 1.27
times more than Trolox. Meanwhile, the peroxyl radical scavenging capacities of the other
compounds were lower than Trolox. The order of antioxidant activity for tested compounds was
TSC-2 > Trolox > TSC-3 > TSC-6 > TSC-4 > TSC-5 > TSC-1, which was similar to the result of the
DPPH assay.
2.2. Theoretical Calculations
During the past few decades, theoretical calculations based on DFT have become a potent
method to elucidate the antioxidant properties of compounds [28,29,37,38]. In order to further
understand the scavenging activity of the tested compounds, the theoretical calculation was
performed at DFT level through the Gaussian 09 software package [39]. The geometry optimizations
and frequency calculations of the investigated species were carried out using the M06-2X/Def2-SVP
level and B3LYP/6-31G(d) level of theory, in conjunction with the polarizable continuum solvation
(PCM) model [40], using ethanol as the solvent.
First at all, the conformational analysis was first performed to identify the most stable
conformational structures of the studied compounds. The optimized structures of the most stable
compounds were shown in Figure 6. The main geometrical parameters of the studied compounds
including bond length and dihedral angle are listed in Table S1 in supporting information, where the
cartesian coordinates of compounds are available.
Figure 5.
Time kinetics plots of
TSC-2
(
A
) and
TSC-3
(
B
) inhibition of non-fluorescent
dichlorofluorescein (DCFH) oxidation by 2,2
0
-azobis(2-amidinopropane) dihydrochloride (AAPH). (the
inset is dose-response plots).
It can be seen that ROO
oxidizes DCFH to fluorescent DCF over time and antioxidants scavenged
peroxyl radicals and inhibited the oxidation reaction in a dose-dependent way. Scavenging peroxyl
radical activity was expressed as EC
50
and as micromoles of Trolox equivalents per micromole of
the test sample. The degree of inhibition was dierent for the investigated compounds, as shown
in Table 1. Among the six compounds,
TSC-2
had the highest PSC value and that indicated that
TSC-2
possess the highest peroxyl radical-scavenging activity, which was 1.27 times more than Trolox.
Meanwhile, the peroxyl radical scavenging capacities of the other compounds were lower than Trolox.
The order of antioxidant activity for tested compounds was
TSC-2 >Trolox >TSC-3 >TSC-6 >TSC-4
>TSC-5 >TSC-1, which was similar to the result of the DPPH assay.
2.2. Theoretical Calculations
During the past few decades, theoretical calculations based on DFT have become a potent method
to elucidate the antioxidant properties of compounds [
28
,
29
,
37
,
38
]. In order to further understand
the scavenging activity of the tested compounds, the theoretical calculation was performed at DFT level
through the Gaussian 09 software package [
39
]. The geometry optimizations and frequency calculations
of the investigated species were carried out using the M06-2X/Def2-SVP level and B3LYP/6-31G(d) level
of theory, in conjunction with the polarizable continuum solvation (PCM) model [
40
], using ethanol as
the solvent.
First at all, the conformational analysis was first performed to identify the most stable
conformational structures of the studied compounds. The optimized structures of the most stable
compounds were shown in Figure 6. The main geometrical parameters of the studied compounds
including bond length and dihedral angle are listed in Table S1 in supporting information, where
the cartesian coordinates of compounds are available.
Molecules 2020,25, 1192 7 of 16
Molecules 2020, 25, x 7 of 15
Figure 6. Geometric structure of the neutral molecules, radical molecules, cation radical molecules
and atomic number of the studied camphene-based thiosemicarbazone. The blue, gray, yellow, white
and red balls represent N atom, .C atom, S atom, H atom and O atom respectively
As shown in Figure 6, the camphene-based thiosemicarbazone structures consisted of a
camphene-based thiosemicarbazide condensed with salicylaldehyde derivations. Individual
differences within the six investigated compounds arise from the property of the substituent. The
Figure 6.
Geometric structure of the neutral molecules, radical molecules, cation radical molecules and
atomic number of the studied camphene-based thiosemicarbazone. The blue, gray, yellow, white and
red balls represent N atom, .C atom, S atom, H atom and O atom respectively.
Molecules 2020,25, 1192 8 of 16
As shown in Figure 6, the camphene-based thiosemicarbazone structures consisted of
a camphene-based thiosemicarbazide condensed with salicylaldehyde derivations. Individual
dierences within the six investigated compounds arise from the property of the substituent.
The geometry is characterized by a dihedral angle of between thiosemicarbazone core moiety and
aromatic ring.
The energy of HOMO and LUMO of a molecule is an important quantum chemical descriptor,
which plays a major role in chemical reactions [
41
]. According to the frontier molecular orbital theory
of chemical reactivity, the formation of the transition state in chemical reactions is due to the interaction
between the HOMO and LUMO orbitals of reacting species. In this work, the frontier orbitals HOMO
and LUMO of the studied compounds were calculated at B3LYP/6-31G(d) level. The energy of
the HOMO-LUMO orbitals of the studied compounds is shown in Table 2and Figure S3. It can be
found that
TSC-2
has an energy gap value of 4.26 eV, the lowest value, indicating that
TSC-2
has
relatively high reactivity.
Table 2.
Rate constants(kb) for radical scavenging, HOMO-LUMO energy gap, bond dissociation
enthalpy (BDE) and adiabatic ionization energy (AIE) values of compounds.
Compounds BDE 1(kcal/mol) kb (M1s1)2HOMO-LUMO
Energy Gap (eV) AIE (eV) 3
NH NH-N=CH OH
TSC-1 98.57 84.63 84.63 16.16 4.53 5.45
TSC-2 98.49 80.86 81.24 4218 4.26 5.41
TSC-3 98.59 85.33 85.92 308.71 4.67 5.49
TSC-4 98.84 86.48 83.53 16.61 4.48 5.56
TSC-5 98.51 86.54 85.47 22.87 4.53 5.56
TSC-6 98.41 85.99 83.51 45.61 4.54 5.46
1
BDE is bond dissociation enthalpy;
2
k
b
represented the bimolecular rate constant, which was determined by
the slope of a linear plot of (dA/dt)0against A0[AH]; represented; 3AIE is adiabatic ionization energy.
In order to evaluate the activity of an antioxidant via the hydrogen donating mechanism, bond
dissociation enthalpy (BDE), which is related to the ability to donate a hydrogen atom, is assessed.
The lowest BDE is defined for the relevant position where the easiest hydrogen atom abstraction for
free radical scavenging reaction can take place [
29
,
42
]. The BDE values of molecules were calculated
by using the B3LYP/6-31G(d) level of theory. There are three possible hydrogen bond breaking sites
in our compounds, and the corresponding BDE values are listed in Table 2. The BDE values of N-H
are in the range of 98.41–98.84 kcal/mol, significantly larger than the BDEs of N-H-N=C and O-H in
molecules (80.86–86.54 kcal/mol). This indicates that the N-H is unfavorable to donating H atoms
compared to the N-H-N=C and O-H groups. It is noteworthy that the calculated N-H-N=C and O-H
BDEs of
TSC-2
are 80.86 and 81.24 kcal/mol, respectively, which are far smaller than other compounds.
This means that
TSC-2
should have a stronger H-donating ability than other compounds; in other
words,
TSC-2
has the highest scavenging radical activity. The calculated result is consistent with
the experimental result.
The optimized structures of the most stable radical for the evaluated compounds are shown
in Figure 6. Table S2 reports the main geometrical parameters of radicals, including bond length
and dihedral angle. It was found that when the H atom was abstracted translating to radical form,
the radical was able to rearrange itself to assume the most stable conformation [
29
]. The obtained
results show that a significant geometrical change in the generated radicals occurs in comparison to
the neutral molecule. For example, in Figure 6, the dihedral angles of
TSC-2
changed from 59.41
to
0.65
—that is, from a non-planar structure to an almost planar structure. The dihedral angle of 0.65
allows a good electronic delocalization, which is favorable for the stability of the radical structure.
The dierence in antioxidant activity was attributed to electron delocalization, which led to
the stabilization of the radicals obtained after the H-atom abstraction. This result is made on
Molecules 2020,25, 1192 9 of 16
the basis of an assumption that if electron delocalization exists in the parent molecule, it also exists
in the corresponding radical. To better understand the relationship between electron delocalization
and the reactivity of the radical, the electron delocalization should be examined. The spin density
distribution oers a better insight into the delocalization of the unpaired electron and conjugation
eects, which is a measure of the stability of the radicals [
43
]. The spin density distribution plot is
presented in Figure 7.
Molecules 2020, 25, x 9 of 15
corresponding radical. To better understand the relationship between electron delocalization and the
reactivity of the radical, the electron delocalization should be examined. The spin density distribution
offers a better insight into the delocalization of the unpaired electron and conjugation effects, which
is a measure of the stability of the radicals [43]. The spin density distribution plot is presented in
Figure 7.
Figure 7. The Spin density distribution of TSC-2 radical.
As can be seen from Figure 7, TSC-2 has more delocalized spin, which stabilizes the radical
formed. These results suggest that the significantly enhanced radical scavenging activity of the
compound TSC-2 is attributed to the stabilization of the radical by both the electron-donating effect
of the diethylamino group and radical delocalization over thiosemicarbazone core moiety and
benzene rings resulting from the almost coplanar structure.
Next, the electron transfer mechanism was analyzed. The concepts of adiabatic ionization energy
(AIE) have been applied successfully in the study of electron transfer reactions. AIE values describe
the electron donation by a better ionization capacity and easier electron donation. The higher the AIE
is, the more difficult an electron is to be removed. Molecules characterized by a low AIE are good
electron donors [28,29]. In this work, the AIE of molecules were calculated using the B3LYP/6-311G(d)
level of theory. The geometrical parameters of cation radicals are available in Table S3. The AIE is
calculated and presented in Table 2. It should be noted that the AIE values have no notable difference,
ranging from 5.41 to 5.56 eV. This means that the ability of electron removal from those compounds
is not very different. In addition, the AIE value is greater than BDE, and hence the mechanism is not
preferred.
In this study, quantum chemical calculation was employed to illuminate TSC-2 with the highest
antioxidant activity on the basis of parameters containing frontier molecular orbitals, BDE, and the
spin density distribution. The results suggest that the hydroxyl group, together with diethylamino
group, play a synergistically antioxidative role for the studied compounds [44].
3. Materials and Methods
All the chemicals and reagents were obtained commercially and used without purification. 6-
hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and 1,1-diphenyl-2-picrylhydrazyl
(DPPH) were purchased from Sigma-Aldrich, Shanghai, China; 2,2-azobis(2-amidinopropane)
dihydrochloride (AAPH) and 2,7-dichlorofluorescin diacetate (DCFH-DA) were supplied by
Macklin, Shanghai, China. Absorbance measurements were recorded on a SHIMASZU UV-2450 UV-
Visible spectrophotometer (Shimadzu Corp., Tokyo, Japan). Fluorescence measurements were
recorded on BioTek Cytation 5 multi-function Microplate Reader (BioTek Instruments, Inc.,
Winooski, VT, USA). 1
H- and 13C-NMR spectra were recorded on a Bruker instrument (Bruker
BioSpin, Rheinstetten, Germany) at 400 and 101MHz, respectively, using DMSO-d6 as solvent.
3.1. Synthesis of the Camphene-Based Thiosemicarbazones (TSC-1~6)
The synthetic pathways and structural features of the compounds are outlined in Scheme 1.
Figure 7. The Spin density distribution of TSC-2 radical.
As can be seen from Figure 7,
TSC-2
has more delocalized spin, which stabilizes the radical formed.
These results suggest that the significantly enhanced radical scavenging activity of the compound
TSC-2
is attributed to the stabilization of the radical by both the electron-donating eect of the diethylamino
group and radical delocalization over thiosemicarbazone core moiety and benzene rings resulting from
the almost coplanar structure.
Next, the electron transfer mechanism was analyzed. The concepts of adiabatic ionization energy
(AIE) have been applied successfully in the study of electron transfer reactions. AIE values describe
the electron donation by a better ionization capacity and easier electron donation. The higher the AIE
is, the more dicult an electron is to be removed. Molecules characterized by a low AIE are good
electron donors [
28
,
29
]. In this work, the AIE of molecules were calculated using the B3LYP/6-311G(d)
level of theory. The geometrical parameters of cation radicals are available in Table S3. The AIE is
calculated and presented in Table 2. It should be noted that the AIE values have no notable dierence,
ranging from 5.41 to 5.56 eV. This means that the ability of electron removal from those compounds
is not very dierent. In addition, the AIE value is greater than BDE, and hence the mechanism is
not preferred.
In this study, quantum chemical calculation was employed to illuminate
TSC-2
with the highest
antioxidant activity on the basis of parameters containing frontier molecular orbitals, BDE, and the spin
density distribution. The results suggest that the hydroxyl group, together with diethylamino group,
play a synergistically antioxidative role for the studied compounds [44].
3. Materials and Methods
All the chemicals and reagents were obtained commercially and used without purification.
6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and 1,1-diphenyl-2-picrylhydrazyl
(DPPH) were purchased from Sigma-Aldrich, Shanghai, China; 2,2
0
-azobis(2-amidinopropane)
dihydrochloride (AAPH) and 2
0
,7
0
-dichlorofluorescin diacetate (DCFH-DA) were supplied by Macklin,
Shanghai, China. Absorbance measurements were recorded on a SHIMASZU UV-2450 UV-Visible
spectrophotometer (Shimadzu Corp., Tokyo, Japan). Fluorescence measurements were recorded on
BioTek Cytation 5 multi-function Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA).
1
H-
and
13
C-NMR spectra were recorded on a Bruker instrument (Bruker BioSpin, Rheinstetten, Germany)
at 400 and 101MHz, respectively, using DMSO-d6 as solvent.
Molecules 2020,25, 1192 10 of 16
3.1. Synthesis of the Camphene-Based Thiosemicarbazones (TSC-1~6)
The synthetic pathways and structural features of the compounds are outlined in Scheme 1.
Molecules 2020, 25, x 10 of 15
Scheme 1. Synthesis and structures of camphene-based thiosemicarbazones.
Compounds 2-isocamphanyl isothiocyanate (1) and 4-(2-isocamphanyl) thiosemicarbazide (2)
were prepared according to the literature procedure [45]. The camphene-based thiosemicarbazones
TSC-1~6 were prepared according to the method described in reference [27]. The synthesized
compounds were characterized by 1H-NMR and 13C-NMR in supporting information.
In 1H-NMR spectra, the thiosemicarbazone moiety contained the imine hydrogen (N=CH) and
two NH. The hydrogen of N=CH is assigned as a singlet at 8.22–8.39 ppm, and the two NH chemical
shifts appeared as two singlets at 11.02–11.45 ppm and 7.78–7.99 ppm. The hydrogen on the phenolic
hydroxyl group was assigned as a singlet at 9.61–10.52 ppm, and aromatic hydrogens appeared at
6.10–7.61 ppm, depending on the substituent in the aromatic group. In the 13C-NMR spectra, the
signals between 97.22 and 175.22 ppm were assigned resonance of aryl and unsaturated carbons. The
most signals at 174.39–175.22 ppm were attributed to the carbon resonance of the thiocarbonyl group
(C=S). The signal for C=N was observed at 136.10–140.07 ppm. All the signals are consistent with the
structures.
3.2. DPPH Radical Scavenging Assay
The DPPH radical scavenging capacity was performed at the fixed reaction time according to
the published method with slight modification [46]. The assay was conducted using a microplate
reader with a spectrophotometric detector.
To ensure complete dissolution, the test compounds were dissolved in a small volume of DMSO
to generate a 10 mM stock solution and diluted to generate the final testing solution with ethanol.
Different concentrations of DPPH solution were prepared to make a linear relationship between
radical concentration and absorbance. The exact DPPH concentration was calculated from the
calibration curve (Figure S4). The DPPH ethanol solution (68 µM) was prepared daily. The 225 µL
ethanol solution of DPPH was added into the 25 µL of tested compounds solution with different
concentrations (or 25 µL ethanol as blank control) on 96-well plates and allowed to react for one hour
at room temperature in the dark. The absorbance was measured at 517 nm on a Microplate Reader.
All tests and analyses were undertaken in three replicates and the results were averaged. The radical
scavenging activity of the evaluated compounds was expressed as the percentage scavenging of free
radical and was calculated using the following equation.
Percentage o
f
Scavenging (%)=(
𝐴
𝐴
)/(
𝐴
𝐴
)] ×100, (1)
Here, A0, A1, and Ablank represent the absorbance of DPPH in the absence or in the presence of
antioxidant and blank.
The effective concentration (EC50) value was defined as the efficient concentration required to
decrease the preliminary DPPH radical concentration by 50%. The value of EC50 was expressed in
terms of the molar ratio of antioxidant to DPPH and estimated by using a dose-response template
from regression models in OriginPro 2019 soft.
3.3. Scavenging DPPH Kinetics Assay
The reaction of DPPH with antioxidants is basically a kinetic driving process. Thus, it is
necessary to assess the kinetic behavior between the DPPH radical and the tested compounds. The
Scheme 1. Synthesis and structures of camphene-based thiosemicarbazones.
Compounds 2-isocamphanyl isothiocyanate (
1
) and 4-(2
0
-isocamphanyl) thiosemicarbazide
(
2
) were prepared according to the literature procedure [
45
]. The camphene-based thiosemicarbazones
TSC-1~6
were prepared according to the method described in reference [
27
]. The synthesized
compounds were characterized by 1H-NMR and 13C-NMR in supporting information.
In
1
H-NMR spectra, the thiosemicarbazone moiety contained the imine hydrogen (N=CH) and
two NH. The hydrogen of N=CH is assigned as a singlet at 8.22–8.39 ppm, and the two NH chemical
shifts appeared as two singlets at 11.02–11.45 ppm and 7.78–7.99 ppm. The hydrogen on the phenolic
hydroxyl group was assigned as a singlet at 9.61–10.52 ppm, and aromatic hydrogens appeared
at 6.10–7.61 ppm, depending on the substituent in the aromatic group. In the
13
C-NMR spectra,
the signals between 97.22 and 175.22 ppm were assigned resonance of aryl and unsaturated carbons.
The most signals at 174.39–175.22 ppm were attributed to the carbon resonance of the thiocarbonyl
group (C=S). The signal for C=N was observed at 136.10–140.07 ppm. All the signals are consistent
with the structures.
3.2. DPPH Radical Scavenging Assay
The DPPH radical scavenging capacity was performed at the fixed reaction time according to
the published method with slight modification [
46
]. The assay was conducted using a microplate
reader with a spectrophotometric detector.
To ensure complete dissolution, the test compounds were dissolved in a small volume of DMSO
to generate a 10 mM stock solution and diluted to generate the final testing solution with ethanol.
Dierent concentrations of DPPH solution were prepared to make a linear relationship between radical
concentration and absorbance. The exact DPPH concentration was calculated from the calibration
curve (Figure S4). The DPPH ethanol solution (68
µ
M) was prepared daily. The 225
µ
L ethanol solution
of DPPH was added into the 25
µ
L of tested compounds solution with dierent concentrations (or
25
µ
L ethanol as blank control) on 96-well plates and allowed to react for one hour at room temperature
in the dark. The absorbance was measured at 517 nm on a Microplate Reader. All tests and analyses
were undertaken in three replicates and the results were averaged. The radical scavenging activity
of the evaluated compounds was expressed as the percentage scavenging of free radical and was
calculated using the following equation.
Percentage of Scavenging (%)=[(A0A1)/(A0Ablank)]×100, (1)
Here, A
0
, A
1
, and A
blank
represent the absorbance of DPPH in the absence or in the presence of
antioxidant and blank.
The eective concentration (EC
50
) value was defined as the ecient concentration required to
decrease the preliminary DPPH radical concentration by 50%. The value of EC
50
was expressed in
Molecules 2020,25, 1192 11 of 16
terms of the molar ratio of antioxidant to DPPH and estimated by using a dose-response template from
regression models in OriginPro 2019 soft.
3.3. Scavenging DPPH Kinetics Assay
The reaction of DPPH with antioxidants is basically a kinetic driving process. Thus, it is necessary
to assess the kinetic behavior between the DPPH radical and the tested compounds. The experiments
were carried out in excess of DPPH radical (the molar ratios of DPPH/antioxidant ranging from
1.5~15:1) in order to use up the H-donating capacity or the e-donating capacity of the tested compounds.
The final concentration of DPPH was about 62
µ
M, while the tested compounds were added in the range
4–40 µM (final concentration).
The DPPH kinetic assay was adopted from the method described previously [
47
]. Briefly, 3900
µ
L
of DPPH solution was added to the 100
µ
L of antioxidant solutions at dierent concentrations (or ethanol
alone as a control). The recording of absorbance at 517nm was initiated immediately. Absorbance was
recorded every 2.5 s for 120 min to generate reaction curves. The delay time between the addition
of DPPH solution and the first absorbance reading was 6 s, and this was considered when plotting
the absorbance–time graph. The decline in absorbance as a function of time is exponential in nature and
plotted for dierent concentrations of antioxidants. The scavenging rate and percentage of remaining
DPPH radical at infinite time were calculated according to the method described by Campos [48].
An empirical bi-exponential [49] Equation (2) was fitted to the date,
y=A1e
x
t1+A2e
x
t2+y0(2)
where the intercept y is constrained to equal A
0
, A
t
is the absorbance at time t; A
1
and A
2
are the fast
decay amplitude and slow decay amplitude, respectively; t
1
and t
2
are time constants of the fast and
slow decays; y0is remaining unreacted DPPH at infinite time.
The bleaching rate is performed by the first derivative of Equation (3),
dA
dt =A1/t1e
x
t1+A2/t2e
x
t2(3)
Substituting x =0 into the below Equation (4) oers an equation for the bleaching rate extrapolated
to zero time,
dA
dt 0=A1k1+A2k2, (4)
A plot of
(dA/dt)0
against A
0
[AH] shows the bimolecular rate constant (k
b
) as the slope of
the linear fit. The extrapolation to “infinite” time was chosen to avoid the uncertainty associated
with the visual estimation of the steady-state conditions [
50
]. The total stoichiometry factor (n) was
calculated according to the previously reported literature [49].
3.4. Peroxyl Radical Scavenging Capacity (PSC) Assay
The total antioxidant activity of the title compounds was determined using the PSC assay
previously described [
35
] with a little modification. Trolox was used as a reference. The tested
compounds and Trolox were diluted by 12% methylated
β
-cyclodextrin in ethanol to six dierent
concentrations. In brief, 100
µ
L of samples or reference were added into a black 96-well microtiter plate,
and then 100
µ
L of DCFH dye (33.06
µ
M) was added. The DCFH dye was prepared by hydrolyzing
DCFH-DA with KOH solution (1 mM) for six min to remove the diacetate moiety before use; later,
50
µ
L of AAPH (40 mM) was added. The reaction proceeded for 36 min at 37
C. Fluorescence
intensity was monitored at the 485 nm excitation wavelength and 538 nm emission wavelength with
a Microplate Reader. The control reactions used 12% methylated
β
-cyclodextrin ethanol solution.
The areas under the average fluorescence–reaction time kinetic curves (AUC) for both the control and
Molecules 2020,25, 1192 12 of 16
the tested compounds were integrated and used to calculate antioxidant activity in accordance with
Equation (5):
PSC unit =1(SA/CA)(5)
where SA and CA represent the AUC for a sample and the control reaction, respectively. The EC
50
was
also expressed as micromoles of Trolox equivalents per micromole of the evaluated compound.
3.5. Theoretical Calculations
According to the previously reported literature [
28
,
30
,
51
], generally, the main mechanisms of
antioxidant action have been proposed and widely accepted, including hydrogen atom transfer (HAT),
single electron transfer (SET) and metal chelation. The DPPH radical and ROO
radical could be
scavenged by the tested compounds via the HAT mechanism or SET mechanism.
In the HAT mechanism, the antioxidant becomes a radical after the free radical removes a hydrogen
atom from the antioxidant. The BDE of N-H band or O-H band is an important parameter in evaluating
the antioxidant action; the lower the BDE value, the easier the dissociation of the N-H or O–H bond. In
the SET mechanism, the AIE is the most significant parameter for the scavenging activity evaluation;
the lower the AIE value, the easier the electron abstraction. It is clear that as far as specific molecular
properties are concerned, BDE and AIE are of particular importance to decide which mechanism is
the favored one [42,52].
In addition, the HOMO-LUMO energy gap value is also an important parameter in determining
the reactivity of a molecule. As the energy gap decreases, the more reactive the molecule will be and
vice versa [41].
Base on the description above, in order to further understand the scavenging activity of the tested
compounds, the theoretical calculation was performed at DFT level. The computational work
was performed through the Gaussian 09 software package [
39
]. The geometry optimizations and
frequency calculations of all the studied species were carried out using the M06-2X/Def2-SVP level
and B3LYP/6-311G(d) level of theory, in conjunction with the polarizable continuum solvation (PCM)
model [
40
] using ethanol as solvent. The frontier orbitals HOMO and LUMO of the studied compounds
and the BDEs of molecules were calculated at the same level used for structural optimization according
to the previously reported literature [
30
,
42
]. The AIEs of the tested compounds were computed using
the B3LYP/6-311G(d) level of theory. Unrestricted calculations were used for the open-shell systems,
such as radicals and radical cation species. To refine the energies, single-point calculations were
performed on the optimized structures at the (U)B3LYP/6-31G(d) level.
All of the electron density analysis was conducted with the help of the Multiwfn software [53].
4. Conclusions
In conclusion, analysis of the antioxidant potential of six camphene-based thiosemicarbazones
was performed by adopting experimental methods including the DPPH assay and the peroxyl
radical scavenging capacity assay. The theoretical approach using DFT was employed to analyze
the experimental results. According to the experimental results, all investigated compounds exhibited
pronounced activity towards DPPH radical and peroxyl radical compared with Trolox. In particular,
TSC-2
exhibited the highest DPPH scavenging activity with the lowest EC
50
(0.208
±
0.004 mol/mole
DPPH), as well as the highest bimolecular rate constant K
b
(M
1
s
1
), which is 1.18 times greater than
Trolox. Meanwhile, in term of scavenging peroxyl radical assay, the experimental data indicated that
TSC-2
also possesses the highest peroxyl racial scavenging activity (1.27
µ
mol of Trolox equiv/
µ
mol).
Furthermore, further explanations for the experimental results have been determined by the DFT
calculation. The analysis of the energy gap of HOMO-LUMO revealed that
TSC-2
has the lowest
energy gap value and suggested that
TSC-2
possesses the best antioxidant activity of the studied
compounds. The lowest BDE for
TSC-2
revealed that
TSC-2
likely acquired a high degree of antiradical
scavenging by the HAT mechanism. The spin density distribution was used to analyze the stabilization
Molecules 2020,25, 1192 13 of 16
of radical formed by HAT. The results suggest that the introduction of a strong electron-donating
group (diethylamino group) in
TSC-2
leads to enhancing the radical scavenging activities by electronic
delocalization. The analysis of the experimental and theoretical data presented in this paper supported
the thesis that
TSC-2
is a promising molecule with high radical scavenging activity. In this work,
the computed parameters gave some useful indication on free radical scavenging behavior of
TSC-2
by H-atom abstraction. In the future work, we need to determine the potential energy surfaces and
the kinetic parameters which give qualitative and quantitative theoretical previsions to elucidate
the antioxidant reaction mechanisms that the TSC-2 compound follows.
Supplementary Materials:
The following are available online, Figure S1: (A)Reaction kinetics of DPPH radical
with TSC-1, TSC-4, TSC-5, TSC-6, and Trolox; (B) %DPPH radical remaining at infinite time at the dierent
concentration of TSC-1, TSC-4, TSC-5, TSC-6 and Trolox, Figure S2: Time kinetics plots of the TSC-1, TSC-4,
TSC-5, TSC-6 inhibition of DCFH oxidation by AAPH. (the inset is dose-response plot); Figure S3: Optimized
ground state structures and the orbital distribution and energy (eV) of HOMO and LUMO for the studied
camphene-based thiosemicarbazone; Figure S4: Calibration curve of DPPH in ethanol at 517 nm; Figure S5~S10:
1H-NMR and 13C-NMR of TSC-1~6; Table S1 Selected bond lengths and dihedral angles of the neutral forms
of thiosemicarbazone in ethanol solution Table S2. Selected bond lengths and dihedral angles of the radicals of
thiosemicarbazone in ethanol solution; Table S3. Selected bond lengths and dihedral angles of the cations radical
of thiosemicarbazone.
Author Contributions:
Experiment, L.Y., H.L.; Calculation, D.X., L.Y.; Writing—Original Draft preparation:
L.Y.; Writing—Review and Editing: L.Y., S.W.; Funding Acquisition, S.W.; All authors have read and approved
the published version of the manuscript.
Funding:
This work was financially supported by the National Natural Science Foundation of China (No. 31470592),
Jiangsu Provincial Key Lab for the Chemistry and Utilization of Agro-Forest Biomass, and Priority Academic
Program Development of Jiangsu Higher Education Institutions, China.
Acknowledgments:
Thanks to Jie Song from University of Michigan-Flint for his guidance in Gaussion calculation.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Tyrosinase plays an essential role in melanogenesis. Tyrosinase inhibition may be not only a means to alleviate skin hyperpigmentation, but also the most effective method for fruit and vegetable preservation. Thiosemicarbazone compounds have received considerable attentions because of their potential therapeutic activities, such as antitumoral, antibacterial and antimalarial properties. In this study, we focused on the screen, toxicity, interaction and inhibitory activities of potent thiosemicarbazone compounds as novel tyrosinase inhibitors. The results showed that the inhibitor effectiveness was drastically reduced by the absence of ortho-hydroxyl group and was improved through changing the substituents of benzaldehyde. To develop a pharmacophore model, we explored the binding mode of thiosemicarbazones compounds in the active site of tyrosinase. Docking analysis using an AUTODOCK program was conducted to explain the flexible thiosemicarbazone molecules binding with the active site of tyrosinase, because of their perfect match with both the active site and the substrate channel of tyrosinase in both size and hydrophobicity. Their binding was stabilized mainly via both the coordination of the sulfur and nitrogen atoms in the inhibitors to binuclear copper in the tyrosinase active site. Further complementary studies on these types of inhibitors, as potential drug candidates for treating abnormal melanin pigmentation and inhibiting food or fruit browning, are still needed.
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In this work the aim of study was the synthesis and evaluation of in vitro anti-Mycobacterium tuberculosis activity as well as the cytotoxicity in VERO cells of a series of 17 novel thiosemicarbazones derived from the natural monoterpene (-)-camphene by REMA and MTT methods. Overall, the majority of tested compounds exhibited considerable inhibitory effects on the growth of M. tuberculosis H37Rv, especially the derivatives 3, 4a–c, 4f, 4i, 4k, 5 and 6a–b. MIC values of 20 tested compounds ranged from 3.9 to > 250 μg/mL. It was found that when inserting new nitrogenous groups to the (-)-camphene increased the anti-M. tuberculosis activity of some compounds. The SI was calculated for all compounds that showed highly potent anti-M. tuberculosis activity and the best SI values were 21.36, 26.92 and 31.62 (4b, 6a and 6b), and may be considered potential candidates for future antituberculosis drugs.
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CuLCl, CuL¹Cl, PdLCl2, and PdL¹Cl2 complexes [L and L¹ being (+)-camphor and (–)-carvone thiosemicarbazones, respectively] have been synthesized. The structure of binuclear [Pd2L²²Cl4] complex has been determined by means of X-ray diffraction. The L² ligand (dehydrogenated (–)-carvone thiosemicarbazone) is coordinated via the bridging S atom to two Pd atoms. The complexes of Cu(I) and Pd(II) presumably have polynuclear and binuclear structure, respectively. These facts are in good agreement with IR and NMR spectroscopy as well as mass spectrometry data which indicate the coordination of L and L¹ ligands via the S atom. The influence of L¹ and PdL¹Cl2 on viability of the Hep2 cell line has been studied. The PdL¹Cl2 complex is more cytotoxic than L¹ ligand.
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A series of 5-methoxysalicylaldehyde appended thiosemicarbazones (1–4) and 2-hydroxy-1-naphthaldehyde appended thiosemicarbazones (5–8) was obtained from the reactions between 5-methoxysalicylaldehyde/2-hydroxy-1-naphthaldehyde and (un)substituted thiosemicarbazides with the view to ascertain their biological properties brought about by the change in substitution at N-terminal position of the thiosemicarbazide derivatives. The compounds were fully characterized by elemental analyses, and various spectroscopic techniques (UV–Visible, FT-IR, NMR and mass). The solid-state structure of three compounds (1, 2 and 7) was determined by single crystal X-ray diffraction method. The compounds (1, 2 and 7) have adopted a monoclinic crystal system with P21/c (1 and 2) or C2/c (7) space group. Antioxidant and non-haemolysis activities of the compounds (1–8) were analyzed by in vitro DPPH and haemolysis assays, respectively. Anti-inflammatory potential was verified by in vitro PLA2 inhibition assay and in silico molecular docking study. In vitro and in silico studies revealed promising anti-inflammtory potential of the thiosemicarbazone derivatives. Compounds 2, 4, 6, 7 and 8 showed significant anti-inflammatory activity.
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A series of new nopinone-based thiosemicarbazone derivatives were designed and synthesized as potent anticancer agents. All these compounds were identified by ¹H-NMR, ¹³C-NMR, HR-MS spectra analyses. In the in vitro anticancer activity, most derivatives showed considerable cytotoxic activity against three human cancer cell lines (MDA-MB-231, SMMC-7721 and Hela). Among them, compound 4i exhibited most potent antitumor activity against three cancer cell lines with the IC50 values of 2.79 ± 0.38, 2.64 ± 0.17 and 3.64 ± 0.13 μM, respectively. Furthermore, the cell cycle analysis indicated that compound 4i caused cell cycle arrest of MDA-MB-231 cells at G2/M phase. The Annexin V-FITC/7-AAD dual staining assay also revealed that compound 4i induced the early apoptosis of MDA-MB-231 cells.