Synthesis, antioxidant and DNA cleavage activities of novel indole derivatives

Article abstract of DOI:10.1016/j.ejmech.2010.05.067

A new series of novel indole derivatives containing barbitone moiety (5a-i) are synthesized by simple and efficient condensation of chalcones (3a-i) with barbituric acid (4). The synthesized compounds are screened for their antioxidant (free radical scave

Full text of DOI:10.1016/j.ejmech.2010.05.067

European Journal of Medicinal Chemistry 45 (2010) 4074e4078
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antioxidant and DNA cleavage activities of novel indole derivatives
*
J.S. Biradar , B.S. Sasidhar, R. Parveen
Central Research Lab, Department of Chemistry, Gulbarga University, Gulbarga, 585106 Karnataka, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of novel indole derivatives containing barbitone moiety (5a-i) are synthesized by simple
and efficient condensation of chalcones (3a-i) with barbituric acid (4). The synthesized compounds are
screened for their antioxidant (free radical scavenging, total antioxidant capacity and ferric reducing
antioxidant power) and DNA cleavage activities were evaluated. Among the synthesized compounds (5a),
(5d) and (5g) exhibited excellent antioxidant activity and all the tested compounds in the series have
exhibited promising DNA cleavage activities. The structures of the synthesized compounds are assigned
on the basis of elemental analysis, IR, 1H NMR, 13C NMR and mass spectral data.
Received 28 October 2009
Received in revised form
26 May 2010
Accepted 28 May 2010
Available online 9 June 2010
Keywords:
Ó 2010 Elsevier Masson SAS. All rights reserved.
Indole-3-carboxaldehyde
VilsmeiereHaack formylation
Barbituric acid
Chalcones
Antioxidant activity
DNA cleavage activity
1. Introduction
many barbituric acid derivatives are known to possess a wide range
of activities, such as hypnotics [19e22]
There is increasing evidence of the implication of free radicals
and reactive oxygen species in a variety of diseases and patho-
physiological events including inflammation, cancer, myocardial
infraction, arthritis and neurodegenerative disorders [1e3]. The
serious consequence of free radical action on biological systems is
a multiple complex aspects of their intervention in a series of
inflammatory [4] and nutritional diseases [5,6]. Many anti-inflam-
matory agents are known to act through the scavenging of oxygen
radicals [7,8]. Deoxyribonucleic acid (DNA) is the primary target
molecule for most anticancer and antiviral therapies [9,10]. The
design of molecules that can bind with DNA provides a formidable
opportunity for chemist with molecules which can offer new
prospects for controlled manipulation of genome.
Indole derivatives constitute an important class of therapeutic
agents in medicinal chemistry including anticancer [11], antioxi-
dant [12], antirheumatoidal and anti-HIV [13,14] and also play
a vital role in the immune system [16,17]. Many indole derivatives
are considered as the most potent scavenger of free radicals [15].
Artificial receptors for biologically active molecules have attracted
attention from the viewpoint of molecular recognition [18].
Derivatives of barbituric acid are important members of the
pyrimidine family, as they are suitable for this purpose. Further
It was envisaged that the two pharmacophores if linked
together (Scheme 1) would generate novel molecular templates
which are likely to exhibit interesting biological properties in
animal models. Owing to the importance and in continuation of our
work on synthesis of biologically active compounds [23e25], we
focused on the investigation of antioxidant properties of pharma-
cologically active compounds and several experimental protocols
have been developed for this purpose and excellent results have
been obtained.
2. Chemistry
A typical synthetic strategy employed to obtain the title
compounds (5aei) in excellent yields is depicted in Scheme 1. In the
present investigation, substituted indole-3-carboxaldehydes (1aec)
were obtained from 2,5-disubstituted indoles formed by Bischler’s
method were subjected to formylation under VilsmeiereHaack
reaction conditions with phosphorous oxychloride and dime-
thylformamide [26]. Various substituted indole-3-carboxaldehydes
(1a-c) were reacted with substituted acetophenones (2aec) in basic
media to get chalcones (3aei) by reported method [27]. The
condensation of chalcones (3aei) with barbituric acid in acetic acid
gave (5aei) [28]. All the newly synthesized compounds were char-
acterized by IR, 1H NMR, 13C NMR and mass spectroscopic data. The
IR spectrum of 5-(3-(1H-indol-3-yl)-1-phenylallylidene) pyrimi-
* Corresponding author. Tel.: þ91 8472 263292; fax: þ91 8472 248632.
E-mail address: jsbiradar@yahoo.com (J.S. Biradar).
dine-2,4,6(1H,3H,5H)-trione (5a) showed
a strong absorption
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.05.067

J.S. Biradar et al. / European Journal of Medicinal Chemistry 45 (2010) 4074e4078
4075
3.1.1.2. Total antioxidant capacity. Total antioxidant activity was
performed to all the newly synthesized compounds [30]. Antioxi-
dant capacities are expressed as equivalents of ascorbic acid.
Among the tested compounds, 5a, 5d and 5g which are simple
indole derivatives have shown very good activity and remaining
compounds shown less activity. The results of total antioxidant
activity were shown in Fig. 2.
H
O
C
R'
H3C
O
C
R"
+
R
N
H
(1a-c)
(2a-c)
1600 C
Piperidine
Ethylene glycol
O
3.1.1.3. Ferric reducing antioxidant power activity. All the novel
compounds were screened for ferric reducing antioxidant activity
[31]. Butylated hydroxy anisole (BHA) was used as standard. Simple
indole derivatives 5a, 5d and 5g have shown more promising
activity and other derivatives have shown moderate activities. The
results are presented in Fig. 3.
R"
R'
R
N
H
(3a-i)
O
HN
O
3.2. DNA cleavage activity
NH
O
The DNA cleavage activity was determined using gel electro-
phoresis by Sambrook et al. [32]. The pictures of the gels are pre-
sented in Fig. 4. The gel after electrophoresis clearly revealed that,
all the tested compounds did act on the DNA as little tailing in the
bands can be observed in treated samples. The difference was
observed in bands of all the compounds compared to the control
DNA. This shows that the control DNA alone does not show any
apparent cleavage as the compounds did. With this, it can be
concluded that the compound inhibits the growth of the patho-
genic organism by cleaving the genome.
R"
O
R'
NH
AcOH
(3a-i)
+
R
O
N
H
O
N
H
(5a-i)
(4)
a
H
H
H
b
c
d
H
H
e
f
g
H
H
h
i
R
R'
R''
Ph Ph
CH3 Cl
Ph Ph
CH3 Cl
Ph Ph
CH3 Cl
4. Conclusion
H
H CH3 CH3 CH3 Cl Cl Cl
Scheme 1. Schematic representation for the synthesis of novel indole derivatives from
barbituric acid.
In conclusion, we have synthesized novel indole derivatives
(5aei) and evaluated these compounds for their antioxidant and
DNA cleavage activities. Most of them demonstrated a broad spec-
trum of antioxidant activities. The simple indole derivatives 5a, 5d
and 5g were concluded as most potent derivatives in all the three
cases. DNA cleavage studies revealed that the test compounds in the
series have exhibited promising cleavage activity. With these
excellent results, it can be concluded that the simple indole and
chlorine at the fifth position of indole moiety enhanced the activity.
Therefore, our findings will provide a great impact on chemists and
biochemists for further investigations in the indole field in search of
molecules possessing potent antioxidant and anticancer activities.
Based on these results, selected novel compounds arebeingscreened
for anticancer activity which will be reported in due course.
at 3408 cmꢀ1 corresponding to indole NH, absorption at 2918 and
2849 cmꢀ1 corresponds to pyrimidine NH/NH, and absorption at
1685 cmꢀ1 corresponds to carbonyl stretching. The 1H NMR spec-
trum of (5a) has exhibited a singlet at
is also D2O exchangeable. Peaks at 10.72 and 10.69 were assigned
to pyrimidine NH/NH and doublet for vicinal protons has merged
with aromatic multiplet between 6.7 and 8.0 ppm. The mass
d 11.19 due to indole NH which
d
d
spectrum of compound 5a has shown molecular ion peak at m/z
359.3 [m þ 2]. The various new indole derivatives synthesized
during the present investigation are listed in Table 1.
3. Results and discussion
3.1. Biological activities
5. Experimental protocols
All the newly synthesized indole derivatives (5aei) are screened
for their biological activities such as antioxidant (free radical
scavenging, total antioxidant capacity and ferric reducing antioxi-
dant power) and DNA cleavage activities were studied by agarose
gel electrophoresis method.
5.1. Chemistry
All the chemicals and reagents were purchased from MERCK,
Himedia and SD Fine Chemical companies and are used without
further purification. Melting points of the synthesized compounds
are determined in open capillaries and are uncorrected. Reactions
are monitored by thin-layer chromatography (TLC) on silica gel 60
F254 aluminium sheets (MERCK). The mobile phase was chloroform
and benzene (1:1) and detection was made using UV light and
iodine. IR spectra are recorded in KBr on PerkineElmer and FTIR
Spectrophotometer (nmax in cmꢀ1). 1H NMR and 13C NMR spectra on
BRUKER AVENE II 400-MHz NMR Spectrometer (Chemical shift in
3.1.1. Antioxidant activities
3.1.1.1. Free radical scavenging activity. The newly synthesized
compounds were screened for free radical scavenging activity by
DPPH method [29]. The samples were prepared at concentrations
of 10, 50, and 100 mg/100 ml, and butylated hydroxy anisole (BHA) is
taken as standard. Simple indole derivatives, 5a, 5d and 5g have
very good scavenging activity. Chloro-substituted indole deriva-
tives 5c, 5f and 5i have shown moderate activities and methyl
derivatives 5b, 5e and 5h have shown least activity compared with
the standard. The bar graph representation of percentage of free
radical scavenging activity is shown in Fig. 1.
d
ppm down field from TMS as an internal reference). The mass
spectra are recorded on LC-MSD-Trap-SL instruments. The
elemental analysis was determined on FLASH EA 1112 SERIES
instrument. All the compounds gave C, H and N analysis within
ꢁ0.5% of the theoretical values.

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J.S. Biradar et al. / European Journal of Medicinal Chemistry 45 (2010) 4074e4078
Table 1
Physical and analytical data of the indole e baritone derivatives.
O
HN
O
NH
O
R"
R'
R
N
H
Entry
Producta
R
R0
R00
Yieldb (%)
m.p.c (ꢂC)
Mol. formula/Mol. Wt.
Elem. Analysis (Cal./Found)
C
H
N
1
2
3
4
5
6
7
8
5a
5b
5c
5d
5e
5f
H
H
H
67
69
57
61
67
55
56
68
67
190e192
195e197
200e203
167e169
210e213
250e252
272e274
243e245
267e270
C21H15N3O3
357
70.58
70.52
4.23
4.26
11.76
11.80
Ph
Ph
H
CH3
Cl
H
C28H21N3O3
447
75.15
75.10
4.73
4.78
09.39
09.43
H
C27H18ClN3O3
467
69.31
69.26
3.88
3.84
08.98
09.07
H
CH3
CH3
CH3
Cl
C22H17N3O3
371
71.15
71.13
4.61
4.64
11.31
11.29
Ph
Ph
H
CH3
Cl
C29H23N3O3
461
75.47
75.50
5.02
5.05
09.10
09.06
C29H20ClN3O3
481
69.78
69.76
4.18
4.20
08.72
08.76
5g
5h
5i
H
C21H14ClN3O3
391
64.37
64.40
3.60
3.54
10.72
10.70
Ph
Ph
CH3
Cl
Cl
C28H20ClN3O3
481
69.78
69.74
4.18
4.21
08.72
08.75
9
Cl
C27H17Cl2N3O3
502
64.55
64.50
3.41
3.45
08.36
08.39
a
Products were characterized by IR, NMR, MS and elemental analysis.
Isolated yields.
Melting points are uncorrected.
b
c
5.1.1. Typical experimental procedure for the synthesis of 2,5-
substituted lindole-3-carboxaldehydes (1aec)
The precursors 2,5-disubstituted indole-3-carboxaldehydes
(1aec) were obtained from the VilsmeiereHaack formylation
reaction of 2,5-disubstituted indoles.
5.1.3. General procedure for the synthesis of (5aei)
To a solution of (3aei) (0.01 mol) in 6e7 mL acetic acid, barbi-
turic acid (4) (0.01 mol) was added. The reaction mixture was then
refluxed for 6e7 h. After completion (TLC) the reaction mixture was
poured into crushed ice with constant stirring. Crude product was
isolated and recrystallized from suitable solvents to yield target
compounds (5aei).
5.1.2. Experimental procedure for the synthesis of 3-(2,5-
substituted-1H-indole-3-yl)-1-phenyl prop-2-en-1-one (3aei)
Various substituted indole-3-carboxaldehydes (1aec) were
reacted with substituted acetophenones (2aec) in ethylene glycol
with catalytic amount of piperidine to yield chalcones (3aei) [27].
5.1.3.1. 5-((E)-3-(1H-indol-3-yl)-1-phenylallylidene)pyrimidine-2,4,6
(1H,3H,5H)-trione (5a). Yield 67% (Benzene): mp 190e192 ꢂC; IR
(KBr) nmax in cmꢀ1: 3408, 2918, 2849 1685, 1589; 1H NMR
Total Antioxidant Capacity Activity
Free Radical Scavenging Activity
80
90
80
70
60
50
40
30
20
10
0
70
60
50
40
30
20
10
0
10 ug
50 ug
100 ug
10 ug
50 ug
100 ug
5a
5b
5c
5d
5e
5f
5g
5h
5i
BHA
5a
5b
5c
5d
5e
5f
5g
5h
5i
BHA
Fig. 2. Total antioxidant capacity of the compounds (5aei).
Fig. 1. Free radical scavenging activity of the synthesized compounds (5aei).

J.S. Biradar et al. / European Journal of Medicinal Chemistry 45 (2010) 4074e4078
4077
CHe); 13C NMR (DMSO-d6 þ CDCl3) in
d
ppm: 163.7 (C]O), 152.0
Ferric Reducing Antioxidant Activity
0.45
0.4
0.35
0.3
0.25
0.2
(C]O), 27.2 (CH3) and 147.0, 134.7, 134, 1131.1, 128.5, 127.8, 127.5,
124.8, 122.1, 117.0 and 114.4; MS: m/z ¼ 372.3 [M þ 1]þ. Anal. Calcd.
for C22H17N3O3: C, 71.15; H, 4.61; N, 11.31%. Found C, 71.13; H, 4.64;
N, 11.29%.
10 ug
50 ug
100 ug
0.15
0.1
0.05
0
5.1.3.5. 5-((E)-3-(5-methyl-2-phenyl-1H-indol-3-yl)-1-p-tolylallyli-
dene)pyrimidine-2,4,6(1H,3H,5H)-trione (5e). Yield 67% (Ethanol):
mp 210e213 ꢂC; IR (KBr) nmax in cmꢀ1: 2957, 2918, 2849, 1697,1591;
5a
5b
5c
5d
5e
5f
5g
5h
5i
BHA
1H NMR (DMSO-d6 þ CDCl3) in
d ppm: 11.10 (s, 1H, indole NH), 9.75
Fig. 3. Ferric reducing antioxidant activity of the compounds (5aei).
(s, 1H, pyrimidine NH), 9.70 (s, 1H, pyrimidine NH), 7.1e8.2 (m, 14H,
12Ar-H, 2CH]CHe), 2.3 (s, 3H, CH3); MS: m/z ¼ 460.5 [M ꢀ 1]þ.
Anal. Calcd. for C29H23N3O3: C, 75.47; H, 5.02; N, 09.10%. Found: C,
75.50; H, 5.05; N, 09.06%.
(DMSO-d6 þ CDCl3) in
d ppm: 11.19 (s, 1H, indole NH), 10.72 (s,
1H, pyrimidine NH), 10.69 (s, 1H, pyrimidine NH), 6.7e8.0 (m,
12H, 10Ar-H, 2CH]CHe); 13C NMR(DMSO-d6 þ CDCl3) in
d ppm:
163.1(C]O), 153.5(C]O) and 146.9, 134.9, 133.8, 131.1, 128.7,
127.8, 125.6, 122.2, 121.7, 120.0 and 115.1; MS: m/z ¼ 359.3
[M þ 2]þ. Anal. Calcd. for C21H15N3O3: C, 70.58; H, 4.23; N,
11.76%. Found: C, 70.52; H, 4.26; N, 11.80%.
5.1.3.6. 5-((E)-3-(5-chloro-2-phenyl-1H-indol-3-yl)-1-p-tolylallyli-
dene)pyrimidine-2,4,6(1H,3H,5H)-trione (5f). Yield 55% (Ethanol):
mp 250e252 ꢂC; IR (KBr) nmax in cmꢀ1: 2959, 2918, 2849, 1684,
1651; 1H NMR (DMSO-d6 þ CDCl3) in
d ppm: 11.01 (s, 1H, indole
NH), 10.21(s, 1H, pyrimidine NH), 10.12 (s, 1H, pyrimidine NH),
7.0e8.1 (m, 14H, 12Ar-H, 2CH]CHe); MS: m/z ¼ 482.4 [M þ 1]þ.
Anal. Calcd. for C29H20ClN3O3: C, 69.78; H, 4.18; N, 08.72%. Found: C,
69.76; H, 4.20; N, 08.76%.
5.1.3.2. 5-((E)-3-(5-methyl-2-phenyl-1H-indol-3-yl)-1-phenyl-
allylidene)pyrimidine-2,4,6(1H,3H,5H)-trione
(5b). Yield
69%
(Benzene): mp 195e197 ꢂC; IR (KBr) nmax in cmꢀ1: 3433, 3051, 2924,
1693, 1599; 1H NMR (DMSO-d6 þ CDCl3) in
d ppm: 11.2 (s, 1H,
indole NH), 9.99 (s, IH, pyrimidine NH), 9.94 (s, IH, pyrimidine NH),
6.8e8.0 (m, 15H, 13Ar-H, 2CH]CHe), 2.4 (s, 3H, CH3); MS: m/
z ¼ 445.4 [M ꢀ 2]þ. Anal. Calcd. for C28H21N3O3: C, 75.15; H, 4.73; N,
09.39%. Found: C, 75.10; H, 4.78; N, 09.43%.
5.1.3.7. 5(E)-1-(4-chlorophenyl)-3-1H-indol-3-yl) allylidene)pyrimi-
dine-2,4,6 (1H,3H,5H)-trione (5g). Yield 56% (Ethanol): mp
272e274 ꢂC; IR (KBr) nmax in cmꢀ1: 3027, 2976, 2849,1684, 1604; 1H
NMR (DMSO-d6 þ CDCl3) in
d ppm: 11.20 (s, 1H, indole NH), 10.20 (s,
1H, pyrimidine NH), 10.19 (s, 1H, pyrimidine NH), 7.1e7.5 (m, 11H,
9Ar-H, 2CH]CHe), 2.2 (s, 3H, CH3); 13C NMR(DMSO-d6 þ CDCl3) in
5.1.3.3. 5-((E)-3-(5-chloro-2-phenyl-1H-indol-3-yl)-1-phenylallyli-
dene)pyrimidine-2,4,6(1H,3H,5H)-trione (5c). Yield 57% (benzene):
mp 200e203 ꢂC; IR (KBr) nmax in cmꢀ1: 3296, 3063, 2916, 1693,
d
ppm: 164.6 (C]O), 155.7 (C]O) and 147.5, 138.9, 137.8, 131.7,
129.3, 128.7, 128.5 124.6, 121.7, 120.7 and 119.3; MS: m/z ¼ 393.1
[M þ 2]þ. Anal. Calcd. for C21H14ClN3O3: C, 64.37; H, 3.60; N, 10.72%.
Found: C, 64.40; H, 3.54; N, 10.70%.
1643; 1H NMR (DMSO-d6 þ CDCl3) in
d ppm: 10.73 (s, 1H, indole
NH), 10.43 (s, 1H, pyrimidine NH), 10.41 (s, 1H, pyrimidine NH),
6.7e8.6 (m, 15H, 13Ar-H, 2CH]CHe); MS: m/z ¼ 469.3 [M þ 2]þ.
Anal. Calcd. for C27H18ClN3O3: C, 69.31; H, 3.88; N, 08.98%. Found: C,
69.26; H, 3.84; N, 09.07%.
5.1.3.8. 5((E)-1-(4-chlorophenyl)-3-(5-methyl-2-phenyl-1H-indol-3-
yl) allylidene) pyrimidine-2,4,6 (1H,3H,5H)-trione (5h). Yield 68%
(Ethanol): mp 243e245 ꢂC; IR (KBr) nmax in cmꢀ1: 2987, 2918, 2849,
1695, 1606; 1H NMR (DMSO-d6 þ CDCl3) in
d ppm: 11.10 (s, 1H,
5.1.3.4. 5-((E)-3-(1H-indol-3-yl)-1-p-tolylallylidene)pyrimidine-
2,4,6(1H,3H,5H)trione (5d). Yield 61% (Ethanol): mp 167e169 ꢂC; IR
(KBr) nmax in cmꢀ1: 3408, 3055, 2918, 1685, 1597; 1H NMR (DMSO-
indole NH), 10.37 (s, 1H, pyrimidine NH), 10.31 (s, 1H, pyrimidine
NH), 7.2e8.2 (m, 14H, 12Ar-H, 2CH]CHe), 2.5 (s, 3H, CH3); MS: m/
z ¼ 482.8 [M þ 1]þ. Anal. Calcd. for C28H20ClN3O3: C, 69.78; H, 4.18;
N, 08.72%. Found: C, 69.74; H, 4.21; N, 08.75%.
d6 þ CDCl3) in
d ppm: 11.59 (s, 1H, indole NH), 9.89 (s, 1H, pyrimi-
dine NH), 9.88 (s,1H, pyrimidine NH), 7.1e8.2 (m,11H, 9Ar-H, 2CH]
Fig. 4. DNA cleavage activity of novel indole derivatives 5a, 5c, 5d, 5f, 5g and 5i.

4078
J.S. Biradar et al. / European Journal of Medicinal Chemistry 45 (2010) 4074e4078
5.1.3.9. 5-((E)-3-(5-chloro-2-phenyl-1H-indol-3-yl)-1-(4-chloro-
phenyl)allylidene) pyrimidine-2,4,6(1H,3H,5H)-trione (5i). Yield 67%
(Ethanol): mp 267e270 ꢂC; IR (KBr) nmax in cmꢀ1: 3227, 2918, 2849,
centrifugation and the pellet was dried and dissolved in TAE buffer
(10 mM tris pH 8.0, 1 mM EDTA) and stored in cold condition.
1716, 1647; 1H NMR (DMSO-d6 þ CDCl3) in
d
ppm: 10.91 (s, IH,
5.3.3. Agarose gel electrophoresis
indole NH), 10.48 (s, 1H, pyrimidine NH), 10.40 (s, 1H, pyrimidine
NH), 7.0e8.2 (m, 14H, 12Ar-H, 2CH]CHe), 2.4 (s, 3H, CH3); MS: m/
z ¼ 502.2 [M ꢀ 1]þ. Anal. Calcd. for C27H17Cl2N3O3: C, 64.55; H, 3.41;
N, 08.36%. Found: C, 64.50; H, 3.45; N, 08.39%.
Cleavage products were analyzed by agarose gel electrophoresis
method [32]. Test samples (1 mg/ml) were prepared in DMF. The
samples (25 mg) were added to the isolated DNA of E. coli. The
samples were incubated for 2 h at 37 ꢂC and then 20 ml of DNA
sample (mixed with bromophenol blue dye at 1:1 ratio) was loaded
carefully into the electrophoresis chamber wells along with stan-
dard DNA marker containing TAE buffer (4.84 g tris base, pH 8.0,
0.5 M EDTA/1 L) and finally loaded on agarose gel and passed the
constant 50 V of electricity for 30 min. Removing the gel and
stained with 10.0 mg/ml ethidium bromide for 10e15 min, the
bands were observed under Vilber Lourmat Gel documentation
system and then photographed to determine the extent of DNA
cleavage. The results are compared with standard DNA marker.
5.2. Biological activities
5.2.1. Antioxidant activities
5.2.1.1. Free radical scavenging activity. Free radical scavenging
activity was done by DPPH method [29]. Different concentrations
(10
(BHA) were taken in different test tubes. The volume was adjusted
to 100 l by adding MeOH. Five milliliters of 0.1 mM methanolic
mg, 50 mg and 100 mg) of samples and butylated hydroxy anisole
m
solution of DPPH was added to these tubes and shaken vigorously.
The tubes were allowed to stand at 27 ꢂC for 20 min. The control
was prepared as above without any extract. The absorbances of
samples were measured at 517 nm. Radical scavenging activity was
calculated using the following formula:
Acknowledgement
One of the authors B.S. Sasidhar is thankful to UGC, New Delhi
110012, India for providing financial assistance through UGC-JRF
(RFSMS) and to BIO GENICS, Dharwad, Karnataka, India for their
assistance in carrying out biological activities.
% Radical scavenging activity ¼ [(Control OD ꢀ Sample OD)/
(Control OD)] ꢃ 100.
5.2.1.2. Total antioxidant capacity. Various concentrations of
extracts (10 mg, 50 mg and 500 mg) were taken in a series of test
References
tubes. To this, 1.9 mL of reagent solution (0.6 M sulfuric acid, 28 mM
sodium phosphate and 4 mM ammonium molybdate) was added.
The tubes were incubated at 95 ꢂC for 90 min and allowed to cool.
The absorbance of each aqueous solution was measured at 695 nm
against a blank. Antioxidant capacities are expressed as equivalents
of ascorbic acid. Ascorbic acid equivalents are calculated using
standard graph of ascorbic acid. The values are expressed as
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5.3. DNA cleavage activity
5.3.1. Preparation of culture media
DNA cleavage experiments were done according to the literature
[32]. Nutrient broth [peptone, 10; yeast extract, 5; NaCl, 10; in (g/l)]
was used for culturing of Escherichia coli. Fifty-milliliter media was
prepared, autoclaved for 15 min at 121 ꢂC under 15 lb pressure. The
autoclaved media were inoculated for 24 h at 37 ꢂC.
5.3.2. Isolation of DNA
The fresh bacterial culture (1.5 ml) is centrifuged to obtain the
pellet which is then dissolved in 0.5 ml of lysis buffer (100 mM tris
pH 8.0, 50 mM EDTA, 10% SDS). To this 0.5 mL of saturated phenol
was added and incubated at 55 ꢂC for 10 min, then centrifuged at
10,000 rpm for 10 min and to the supernatant, equal volume of
chloroform: isoamyl alcohol (24:1) and 1/20th volume of 3 M
sodium acetate (pH 4.8) was added. Centrifuging at 10,000 rpm for
10 min and to the supernatant, 3 volumes of chilled absolute
alcohol were added. The precipitated DNA was separated by
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