S1
Antioxidant and antimicrobial assessment of synthesized and spectrally
characterized new nitrophenothiazines and their sulfone analogues
Naveen Gautam1, Ankita Garg*2, Ajay Kumar Bishnoi2, Shikha Agarwal3, Dinesh
Chand Gautam2
1
2
Government P.G. College, Kotputli, Jaipur 303108
Department of Chemistry, University of Rajasthan, Jaipur 302004
3
MLS University, Udaipur, 313001, India
Email: garg_ankita01@yahoo.com
Supplemental Materials
Biological Activity of the synthesized compounds
Antioxidant Activity
The synthesized compounds were screened for antioxidant activity by 1,1-diphenyl-2picrylhydrazyl (DPPH) radical scavenging assay and 2,2-azinobis(3-ethylbenzothiazoline-6sulfonic acid) (ABTS●+) radical cation decolorization assay. The results are tabulated in Tables
S 1 and S 2.
DPPH Radical Scavenging Assay
Radical
scavenging
activity
of
all
synthesized
compounds
was
determined
spectrophotometrically against stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical by
Cuendet et al39.
ABTS Radical Cation Decolorization Assay
The (ABTS●+) assay was carried out using the improved assay of Re et al.40, which is
based on the oxidation of ABTS with potassium persulfate leading to (ABTS●+).
S2
Results and Discussion
All the synthesized compounds were screened for their antioxidant activity by 1,1diphenyl-2-picryl
hydrazyl
(DPPH)
radial
scavenging
assay and
2,2´-azinobis(3-
ethylbenzothiazoline-6-sulfonic acid) (ABTS.+) radical cation decolorization assay.
The present study41-43 demonstrated that the synthesized compounds showed mixed
radical scavenging activity in both DPPH and ABTS assays.
(a) Those compounds showed strong radical scavenging activity in DPPH assay that have
DPPH% inhibition > 50.
(b) Those compounds showed moderate radical scavenging activity in DPPH assay that
have DPPH% inhibition > 30.
(c) Those compounds showed mild radical scavenging activity in DPPH assay that have
DPPH% inhibition < 30.
(d) Those compounds were found to be more active in ABTS assay which showed activity
near to standard compound.
Antimicrobial Activity
Antibacterial Activity :
In this method, paper disc impregnated with compounds dissolved in solvent DMF at
concentrations 25, 50 and 100 μg mL-1. Then the disc impregnated with the solution was placed
on the surface of the media inoculated with the bacterial strain. The plates were incubated at
35°C for 24 h for bacterial cultures. After incubation, the zones inhibition around the disc were
observed. Each testing is done in triplicate. Ciprofloxacin/ Ampicillin sulfate at conc. 50 μg
mL-1 was used as standard drug for antibacterial activity. results were interpreted in terms of
diameter (mm) of zone of inhibition. The % Activity Index for the complex was calculated by
the formula as under:
S3
% Activity Index=
Zone of inhibition by test compound (diameter)
 100
Zone of inhibition by standard (diameter)
Antifungal activity
Antifungal activity of synthesized compounds was tested on fungal strains: A. niger,
C. albicans, Aspergillus solani MTCC 2101, Fusarium culmorum MTCC 349 using disc
diffusion method. In the disc-diffusion method, disc impregnated with compounds dissolved
in solvent DMF at concentrations 25, 50 and 100 μg mL-1 were spread over microorganism
culture in nutrient agar medium. The plates were incubated at 25°C for 48 h for fungal strains.
After incubation the growth inhibiting zones around the disc was observed. Growth inhibiting
zone indicates that the compounds inhibit growth of microorganism. Each experiment is done
in triplicate. Griseofulvin/ fluconazole at concentration 50 μgmL-1 was used as standard drug
for antifungal activity. Results were interpreted in terms of diameter (mm) of zone of inhibition.
The percentage inhibition was calculated by the following equation.
% Inhibition = (C–T) 100/C
Where, C and T are the diameters of the fungal colony in the control and the test plates,
respectively.
Minimum Inhibitory Concentrations:
Minimum inhibitory concentrations (MICs) are defined as the lowest concentration of
antimicrobials that inhibit the visible growth of a microorganism after overnight incubation at
37°C. Determination of the MIC is a semiquantitative test, which gives an approximate idea of
the least concentration of an antimicrobial (test) solution needed to parent microbial growth.
The MIC was determined by the liquid dilution method. Gram positive bacteria
(Staphylococcus aureus, Bacillus subtilis, Micromonospora sp. MTCC 3296) and gram
negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Zymomonas mobilis MTCC 88)
were used as quality control strains. For the antifungal activities of the compounds Candida
S4
albicans, Aspergillus niger, Aspergillus solani MTCC 2101, Fusarium culmorum MTCC 349
were tested. Ciprofloxacin/ Ampicillin sulfate and Griseofulvin/ fluconazole were used as
standard antibacterial and antifungal agents. The stock solutions of test compounds with 1 to
20 μg/mL concentrations were prepared with aqueous methanol. Inoculums of the overnight
culture were prepared. In a series of tubes, 1 mL each of stock solutions of test compound with
different concentrations was taken and 0.4 mL of the inoculums was added to each tube. Further
4.0 mL of sterile water was added to each of the test tubes. These test tubes were incubated for
22-24 h and observed for the presence of turbidity. The absorbance of the suspension of the
inoculums was observed with spectrophotometer at 555 nm. The end result of the test was the
minimum concentration of antimicrobial (test) solutions, which gave clear solution, i.e. no
visual growth. The activity indices of tested compounds against certain bacteria and fungi were
calculated and the results are tabulated in Tables S 3 - S 6.
S5
Table S 1 : Antioxidant activity of synthesized substituted nitrophenothiazines 3c-e
Compounds
DPPH% Inhibitory
(1mg/mL)
ABTS. + activity at different time intervals
0 min.
3c
3d
3e
Ascorbic acid
39.77±0.50
52.03±1.2
27.12±1.5
-
0.690
0.697
0.689
0.694
1 min.
0.503
0.256
0.679
0.040
2 min.
0.155
0.142
0.392
0.003
4 min.
6 min.
0.141
0.118
0.219
0.003
0.112
0.103
0.150
0.003
4 min.
6 min.
Ascorbic acid is used as reference compound whose DPPH % inhibition of 1 mg/ml is 92.96%
Table S 2 : Antioxidant activity of synthesized substituted nitrophenothiazine sufones 4c-e
Compounds
DPPH% Inhibitory
(1mg/mL)
ABTS. + activity at different time intervals
0 min.
4c
4d
4e
Ascorbic acid
48.84±0.01
31.15±0.10
48.59±0.09
-
0.691
0.717
0.692
0.694
1 min.
0.444
0.569
0.242
0.040
Ascorbic acid is used as reference compound whose DPPH % inhibition of 1 mg/ml is 92.96%
2 min.
0.099
0.396
0.110
0.003
0.082
0.333
0.100
0.003
0.070
0.233
0.095
0.003
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Table S 3: Antimicrobial activity of synthesized substituted nitrophenothiazines 3a-b
Micromonospora sp. MTCC 3296
Compounds
IZ
AI
(cm)
Zymomonas mobilis MTCC 88
MIC
IZ
g/mL)
(cm)
AI
Aspergillus solani MTCC 2101
MIC
IZ
g/mL)
(cm)
AI
Fusarium culmorum MTCC 349
MIC
IZ
g/mL)
(cm)
AI
MIC
g/mL)
3ª
2.4
0.857
30.0
2.2
1.047
21.0
2.0
0.666
21.5
2.3
0.851
22.0
3b
1.4
0.5
34.5
1.8
0.857
24.0
2.1
0.7
21.0
1.4
0.518
27.5
Ampicillin
sulfate
2.8
--
28.0
2.1
--
22.0
--
--
--
--
--
--
--
--
--
--
--
3.0
--
18.0
2.7
--
20.0
Fluconazole
Ampicillin sulfate and Flucanozol were used as standard antibacterial and antifungal drugs respectively.
IZ : Inhibition Zone (cm)
AI : Activation Index
MIC : Minimum Inhibition Concentration g/mL)
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Table S 4: Antimicrobial activity of synthesized substituted nitrophenothiazine sulfones 4a-b
Compounds
Micromonospora sp. MTCC 3296
IZ
AI
(cm)
Zymomonas mobilis MTCC 88
MIC
IZ
g/mL)
(cm)
AI
Aspergillus solani MTCC 2101
MIC
IZ
g/mL)
(cm)
AI
Fusarium culmorum MTCC 349
MIC
IZ
g/mL)
(cm)
AI
MIC
g/mL)
4ª
2.7
0.964
28.5
2.0
0.952
23.0
2.2
0.733
20.5
2.1
0.777
23.0
4b
2.6
0.928
29.0
2.2
1.047
21.0
2.1
0.7
21.0
1.5
0.555
27.0
Ampicillin
sulfate
2.8
--
28.0
2.1
--
22.0
--
--
--
--
--
--
Fluconazole
--
--
--
--
--
--
3.0
--
18.0
2.7
--
20.0
Ampicillin sulfate and Flucanozol were used as standard antibacterial and antifungal drugs respectively.
IZ : Inhibition Zone (cm)
AI : Activation Index
MIC : Minimum Inhibition Concentration g/mL)
S8
Table S 5 Antimicrobial activity of synthesized substituted nitrophenothiazines 3c-e.
Compounds
E. coli
B. subtilis
P. aeruginosa
S. aureus
C. albicans
A. niger
MIC
(g/mL) of
bacterial
strain
%AI
(100
g/mL
)
MIC
(g/mL) of
bacterial
strain
%AI
(100
g/mL
)
MIC
(g/mL) of
bacterial
strain
%AI
(100
g/mL
)
MIC
(g/mL) of
bacterial
strain
%AI
(100
g/mL
)
MIC
(g/mL) of
bacterial
strain
%I
(100
g/mL
)
MIC
(g/mL) of
bacterial
strain
%I
(100
g/mL
)
3c
9.66±0.20
67
9.09±0.11
59
8.14±0.29
64
9.93±0.13
63
9.99±0.19
68
8.88±0.23
70
3d
8.20±0.17
64
9.01±0.21
59
8.72±0.16
67
8.07±0.11
69
10.71±0.39
39
11.48±0.25
45
3e
11.68±0.11
73
12.32±0.12
60
12.11±0.26
60
11.39±0.20
62
11.82±0.17
70
13.31±0.17
72
Ciprofloxacin
04.10±0.10
100
04.90±0.13
100
03.85±0.15
100
04.90±0.11
100
-
-
-
-
Griseofulvin
-
-
-
-
-
-
-
-
03.10±0.80
100
04.80±0.10
100
MIC, Minimum Inhibitory Concentration; AI, Activity Index; I, Inhibition
S9
Table S 6 Antimicrobial activity of synthesized substituted nitrophenothiazine sulfones 4c-e.
Compounds
E. coli
B. subtilis
P. aeruginosa
MIC
(g/mL) of
bacterial
strain
%AI
(100
g/mL
)
MIC
(g/mL) of
bacterial
strain
%AI
g/mL
4c
8.43±0.21
67
9.11±0.16
4d
9.67±0.19
74
4e
8.19±0.14
Ciprofloxacin
Griseofulvin
S. aureus
C. albicans
A. niger
MIC
(g/mL) of
bacterial
strain
%AI
(100
g/mL
)
MIC
(g/mL) of
bacterial
strain
%AI
(100
g/mL
)
MIC
(g/mL) of
bacterial
strain
%I
(100
g/mL
)
MIC
(g/mL) of
bacterial
strain
%I
(100
g/mL
)
50
10.22±0.16
64
9.23±0.14
63
10.68±0.48
68
11.34±0.17
70
10.13±0.17
63
9.44±0.22
67
10.44±0.21
69
10.11±0.18
39
9.86±0.14
45
75
8.71±0.20
58
8.00±0.18
60
8.64±0.18
62
9.62±0.32
70
8.24±0.22
72
04.10±0.10
100
04.90±0.13
100
03.85±0.15
100
04.90±0.11
100
-
-
-
-
-
-
-
-
-
-
-
-
03.10±0.80
100
04.80±0.10
100
(100
)
MIC, Minimum Inhibitory Concentration; AI, Activity Index; I, Inhibition
S 10
Figure S 1: 1H NMR of 3a