Photogeneration of reactive oxygen species and photoinduced plasmid DNA cleavage by novel synthetic chalcones

Article abstract of DOI:10.1016/j.jphotobiol.2010.12.004

This paper describes the synthesis and photodynamic properties of six different chalcone derivatives. Using N,N-dimethyl-4-nitrosoaniline (RNO) bleaching assay, the singlet oxygen generating efficiencies of these chalcones are determined relative to rose bengal (RB). Superoxide dismutase (SOD) inhibitable cytochrome c reduction assay and electron magnetic resonance (EMR) spin trapping techniques are used to determine the superoxide anion radical (O2-) yield upon photoirradiation. Photoinduced DNA scission studies show that O2- is involved in the DNA strand break. In addition, antimicrobial activity of these chalcones is also investigated. Structure activity relationship accounts for the difference in the photogeneration of reactive oxygen species (ROS) by these sensitizers. Presence of electron releasing -OCH₃ groups enhances the photogeneration of ROS. Cyclic voltammetry studies indicate a correlation between enzymatic O2- generation efficiency and redox potential of chalcones. Both O2- (Type I) and ¹O₂ (Type II) paths are involved in the photosensitization of chalcones. The LUMO energies obtained by molecular modeling correlate with the one-electron reduction potentials.

Full text of DOI:10.1016/j.jphotobiol.2010.12.004

Journal of Photochemistry and Photobiology B: Biology 102 (2011) 200–208
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
Photogeneration of reactive oxygen species and photoinduced plasmid DNA
cleavage by novel synthetic chalcones
Y. Yesuthangam a, S. Pandian b, K. Venkatesan b, R. Gandhidasan b, R. Murugesan b,c,
⇑
a Department of Chemistry, Jayaraj Annapackiam College for Women, Periyakulam, Theni 625 601, Tamilnadu, India
b School of Chemistry, Madurai Kamaraj University, Madurai 625 021, Tamilnadu, India
c School of Biological Sciences, Madurai Kamaraj University, Madurai 625 021, Tamilnadu, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 June 2010
Received in revised form 25 October 2010
Accepted 3 December 2010
Available online 8 December 2010
This paper describes the synthesis and photodynamic properties of six different chalcone derivatives.
Using N,N-dimethyl-4-nitrosoaniline (RNO) bleaching assay, the singlet oxygen generating efficiencies
of these chalcones are determined relative to rose bengal (RB). Superoxide dismutase (SOD) inhibitable
cytochrome c reduction assay and electron magnetic resonance (EMR) spin trapping techniques are used
to determine the superoxide anion radical (OÅ2ꢀ) yield upon photoirradiation. Photoinduced DNA scission
studies show that OÅ2ꢀ is involved in the DNA strand break. In addition, antimicrobial activity of these
chalcones is also investigated. Structure activity relationship accounts for the difference in the photogen-
eration of reactive oxygen species (ROS) by these sensitizers. Presence of electron releasing –OCH3 groups
enhances the photogeneration of ROS. Cyclic voltammetry studies indicate a correlation between enzy-
matic OÅ2ꢀ generation efficiency and redox potential of chalcones. Both O2Åꢀ (Type I) and 1O2 (Type II) paths
are involved in the photosensitization of chalcones. The LUMO energies obtained by molecular modeling
correlate with the one-electron reduction potentials.
Keywords:
Chalcones
Spin trapping
Singlet oxygen
Superoxide anion
DNA cleavage
Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction
find various applications as photosensitive materials, including
second harmonic generation materials in non-linear optics [17],
Chalcones, bichromophoric molecules, separated by a keto-
vinyl chain, constitute an important class of naturally occurring
flavonoids and exhibit a wide spectrum of biological activities
[1–5]. For example, chalcones, isolated from the exudates of
Angelica keiskei, are found to exhibit chemopreventive and anti-
metastatic effects [6–9]. Chalcone isoliquiritigenin, from licorice,
exhibits antitumerigenic activity [10]. Licochalcone is reported to
show significant antitumor activity in various malignant human
cell lines [11]. The simple unsubstituted chalcone itself is shown
to inhibit the proliferation of human breast cancer cells by induc-
ing apoptosis and blocking cell cycle progression [12]. Hydroxy-
chalcones are found to induce apoptosis in B16-F10 melanoma
cells via GSH and ATP depletion [13]. Recently, it is reported that
butein (3,4,20,40-tetrahydroxychalcone), exhibits antiproliferative
effects against tumor cells through suppression of the signal trans-
ducer and activator of transcription 3 (STAT3) pathway [14].
Chalcones also form a new and interesting class of compounds
that are known to be as effective photosensitive materials [15].
These bichromophoric systems are good models for study of the
photoinduced electron transfer process [16]. In addition, chalcones
photorefractive polymers [18], holographic recording materials
[19] and fluorescent probes for sensing of metal ions [20].
Although the chalcone moiety possesses high photoreactivity
[21], studies on the photogeneration of ROS by chalcones are very
limited. Here we present the synthesis of six new chalcones, CH1,
CH2, CH3, CH4, CH5 and CH6 and report their photodynamic action
to generate ROS, investigated using optical and electron magnetic
resonance (EMR) spin trapping techniques. These chalcones
differ in number and position of methoxy substituents. To under-
stand the substituent effects on the generation of ROS, molecular
modeling and cyclic voltammetry studies are done. In addition,
antimicrobial activity and photoinduced DNA cleavage of these
chalcones are also investigated.
2. Materials and methods
2.1. Chemicals
Superoxide dismutase (Cu,Zn-SOD), catalase and cytochrome c
were purchased from Sigma Chemical Co., while reduced nicotin-
amide adenine dinucleotide (NADH) was obtained from Boehringer
Mannheim. N,N-dimethyl-4-nitrosoaniline (RNO), 1,4-diazabicy-
clo[2,2,2]octane (DABCO), diethyltriaminopentaacetic acid (DETA-
PAC), and rose bengal (RB) were obtained from Aldrich. The spin
⇑
Corresponding author. Address: School of Biological Sciences, Madurai Kamaraj
University, Madurai 625 021, India. Tel./fax: +91 452 2459873.
E-mail address: rammurugesan@yahoo.com (R. Murugesan).
1011-1344/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.jphotobiol.2010.12.004

Y. Yesuthangam et al. / Journal of Photochemistry and Photobiology B: Biology 102 (2011) 200–208
201
trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was obtained from
2.3.5. 1-(20-Hydroxy-30,40,60-trimethoxyphenyl)-3-(1H-indol-3-yl)-2-
Aldrich and was purified by activated charcoal method [22].
2,2,6,6-Tetramethyl piperidinol (TEMPL) was obtained from Merck,
India. Imidazole, ethylenediamine-tetraacetic acid (EDTA) and so-
dium azide were purchased from S.D. Fine Chemicals, India. In-
dole-3-aldehyde was obtained from SISCO research laboratories,
India. Imidazole was used after repeated crystallisation from dou-
bly distilled water. All other compounds were used as received.
propen-1-one (CH5)
Yellow coloured solid M.P. 200–202 °C. kamcaextone: 502 nm.
m
:
KBr
max
3043 (OH), and 1637 (>C@O) cmꢀ1 1H NMR (CDCl3): d, ppm 12.2
.
(s, 1H, 20-OH), 9.90 (s, 1H, >NH), 3.80 (s, 9H, 30,40,60-OCH3), 8.19
(d, 1H, b-H, J = 15.3 Hz), 7.49 (d, 1H,
a-H, J = 15.3 Hz), 7.19 (s, 1H,
50-H), 7.26 (m, 2H, 5,6-H), 7.99 (m, 4H, 4,5,6,7-H).
2.3.6. 1-(20-Hydroxy-40,60-dimethoxyphenyl)-3-(1H-indol-3-yl)-2-
propen-1-one (CH6)
2.2. Synthesis of chalcones
Yellow coloured solid M.P. 196–198 °C. kamcaextone: 402 nm.
m
:
KBr
max
3110 (OH), and 1610 (>C@O) cmꢀ1 1H NMR (CDCl3): d, ppm
.
Chalcones were synthesized by condensing different o-hydroxy-
acetophenones with indole-3-aldehydes in the presence of piperi-
dine. In a typical experiment, for example, a mixture containing
2-hydroxyacetophenone and indole-3-aldehyde (in the ratio of
1.0:1.10) in ethanol solution was refluxed in the presence of piper-
idine till the reaction was completed. The reaction was monitored
by thin layer chromatography (TLC) and at the end of the reaction
ethanol was evaporated from the reaction mixture and the resulting
residue was treated with ice-water. After keeping overnight in the
refrigerator, the resulting solid was filtered and crystallized from
ethanol. In each case, the chalcone obtained was characterized by
UV, IR and NMR spectral techniques.
14.05 (s, 1H, 20-OH), 8.99 (s, 1H, >NH), 3.82 (s, 3H, 40-OCH3), 3.87
(s, 3H, 60-OCH3), 7.73 (d, 1H,
a-H, J = 15.3 Hz), 8.15 (d, 1H, b-H,
J = 15.3 Hz), 6.00 (d, 1H, 30-H, J = 2.4 Hz), 6.12 (d, 1H, 50-H,
J = 2.4 Hz), 7.45 (d, 1H, 2-H, J = 2.1 Hz), 8.12 (m, 1H, 4-H), 7.43
(m, 1H, 7-H), 7.38 (m, 2H, 5,6-H).
2.4. Light source
Light source used for irradiation was a 150 W xenon lamp. A fil-
ter combination of 10 cm potassium iodide solution (1 g in 100 ml)
and 1 cm pyridine was used to cut off below 300 nm and to achieve
a spectral window of 300–700 nm. The reaction mixture in a quartz
cuvette, placed at a distance of 12 cm from the light source was
continuously stirred during irradiation.
2.3. Characterization of chalcones
2.3.1. 1-(20-Hydroxyphenyl)-3-(1H-indol-3-yl)-2-propen-1-one (CH1)
Yellow crystalline solid M.P. 165 °C. kamcaextone: 219 and 400 nm.
2.5. Singlet oxygen detection
ꢀ1
KBr
max
m
: 3295 (OH), 2921 (>NAH) and 1631 (>C@O) cm
.
1H NMR
(CDCl3): d, ppm 13.5 (s, 1H, 20-OH), 8.72 (s, 1H, >NH), 7.65 (d, 1H,
2-H; J = 2.1 Hz), 7.71 (d, 1H, -H; J = 15.3 Hz), 8.22 (d, 1H, b-H;
2.5.1. Optical method
Generation of 1O2 by photosensitization under aerobic condi-
tion was measured by RNO bleaching method as described in liter-
ature [23,24]. Optical measurements were made using Shimadzu
UV–VIS spectrometer (UV-160) or Specord S 100 UV–VIS spec-
trometer, Analytik Jena AG, Jena, Germany. The sensitizers were
exposed to light in the presence of imidazole (10 mM) and RNO
(50 mM) in phosphate buffer (pH = 7.4). Additional support for
the formation of 1O2 was obtained by carrying out the reaction in
the presence of 1O2 quenchers and following the inhibition of
RNO bleaching. The rate of disappearance of quencher (A) obeys
the following equation:
a
J = 15.3 Hz), 6.92 (d, 1H, 50-H; J = 8.1, 7.5, 1.2 Hz), 7.04 (d, 1H,
6-H; J = 8.4, 1.2 Hz), 7.98 (d, 1H, 60-H; J = 8.1, 1.2 Hz), 7.49 (m,
1H, 40-H), 7.46 (m, 1H, 7-H), 8.05 (d, 1H, 4-H; J = 4.2 Hz), 7.36
(m, 2H, 5,6-H).
2.3.2. 1-(20-Hydroxy-40-methoxyphenyl)-3-(1H-indol-3-yl)-2-propen-
1-one (CH2)
Yellow coloured solid M.P. 195 °C. kamcaextone: 219 aꢀn1d 396 nm.
KBr
max
m
: 3374 (OH), 2965 (>NAH) and 1625 (>C@O) cm .
1H NMR
(CDCl3): d, ppm 13.9 (s, 1H, 20-OH), 3.87 (s, 3H, OCH3), 8.60 (s,
1H, >NH), 7.65 (d, 1H, 2-H; J = 2.1 Hz), 8.18 (d, 1H, b-H; J =
d½Aꢁ ðIabU1O2ÞKr½Aꢁ
15.3 Hz), 7.80 (d, 1H,
a
-H; J = 15.3 Hz), 6.54 (d, 1H, 30-H;
ꢀ
¼
dt
Kd
J = 2.4 Hz), 6.44 (dd, 1H, 50-H; J = 11.4, 2.1 Hz), 7.95 (d, 1H, 60-H;
J = 9.9 Hz), 7.46 (d, 1H, 7-H), 8.04 (d, 1H, 4-H), 7.33 (m, 2H, 5,6-H).
where Kr is the rate constant for chemical quenching of 1O2 by A, Kd
is the rate constant for deactivation of 1O2 by solvent and Iab is the
intensity of light absorbed by the sensitizer. Each compound under
investigation along with the reference singlet oxygen generator RB,
was studied under identical conditions. The relative ratio of the
slopes of the chalcones and RB, after correction for molar absorption
and photon energy [25] was used to compute the relative efficiency
2.3.3. 1-(20-Hydroxy-50-methoxyphenyl)-3-(1H-indol-3-yl)-2-propen-
1-one (CH3)
Yellow coloured solid M.P. 201 °C. kamcaextone: 408 nm.
m
: 3444
max
KBr
(OH), 3166 (>NAH) and 1633 (>C@O) cmꢀ1
.
1H NMR (CDCl3): d,
ppm 12.8 (s, 1H, 20-OH), 3.87 (s, 3H, OCH3), 8.60 (s, 1H, >NH),
of singlet oxygen generation taking
U
(1O2) = 0.76 for RB [26].
8.18 (d, 1H, b-H, J = 15.3 Hz), 7.64 (d, 1H,
a-H, J = 15.3 Hz), 7.53
Contribution of OÅ2ꢀ and H2O2 in the RNO bleaching was eliminated
by the addition of SOD and catalase.
(d, 1H, 2-H, J = 2.1 Hz), 6.97 (d, 1H, 30-H, J = 9.0 Hz), 7.11 (m, 1H,
40-H), 7.97 (m, 1H, 60-H), 7.45 (m, 1H, 7-H), 7.34 (m, 2H, 5,6-H),
8.03 (m, 1H, 4-H).
2.5.2. EMR-TEMPL method
2.3.4. 1-(20-Hydroxy-30,40-dimethoxyphenyl)-3-(1H-indol-3-yl)-2-
A JEOL JES-TE100 ESR spectrometer was used for EMR measure-
ments. The photoproduction of 1O2 by chalcones can be readily
studied by EMR spectroscopy using TEMPL as singlet oxygen probe
[27]. Reaction mixture (1 ml) containing 0.01 M TEMPL and
0.2 mM chalcone in DMSO was irradiated. The reaction mixture
propen-1-one (CH4)
Yellow coloured solid M.P. 212–214 °C. kamcaextone: 395 nm.
m
:
max
KBr
3438 (OH), and 1627 (>C@O) cmꢀ1
.
1H NMR (CDCl3): d, ppm 13.6
(s, 1H, 20-OH), 3.94 (s, 3H, 30-OCH3), 3.96 (s, 3H, 40-OCH3), 8.70 (s,
1H, >NH), 8.15 (d, 1H, b-H, J = 15.3 Hz), 7.42 (d, 1H,
a
-H,
(100 lM) was drawn into gas-permeable Teflon capillary tube
J = 15.3 Hz), 6.56 (d, 1H, 50-H, J = 9.0 Hz), 7.97 (d, 1H, 60-H,
J = 9.0 Hz), 7.61 (d, 1H, 2-H), 7.49 (m, 1H, 7-H), 8.33 (m, 1H, 4-H),
8.04 (m, 2H, 5,6-H).
(0.8 mm side diameter, 0.5 mm wall thickness) which was folded
and inserted into a narrow quartz tube and placed in the EMR cav-
ity for measurements. The increase in EMR signal intensity of the

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Y. Yesuthangam et al. / Journal of Photochemistry and Photobiology B: Biology 102 (2011) 200–208
2,2,6,6-tetramethyl-4-piperidinol-N-oxyl (TEMPOL) radical pro-
duced was followed.
and the antifungal activity was measured as the zone of inhibition
against yeast.
2.6. Superoxide anion radical detection
2.9. Photoinduced DNA cleavage by chalcones
2.6.1. Optical method
An in vitro assay for DNA strand breaks, induced by ROS de-
pends on the migration rates of supercoiled (unnicked), relaxed
circular (nicked), linear and degraded plasmid DNA in agarose gel
electrophoresis. Plasmid DNA (Genetix Biotech Asia Pvt. Ltd.)
The generation of superoxide anion radical was detected by the
reduction of Fe(III)-Cyt.c by OÅ2ꢀ. Solutions of chalcones (100
l
M)
M) in
50 mM phosphate buffer (pH = 7.4). The generation of ferrocyto-
chrome was monitored spectrophotometrically at 550 nm
using
OD550 = 20,000 Mꢀ1 cmꢀ1 for reduced-oxidised cytochrome
were photolysed in the presence of ferricytochrome c (40
l
(3
phate buffer (pH = 7.4) was irradiated for 10 min in gas-permeable
Teflon capillary tube. After the irradiation was over, 20 l aliquot of
the mixture was loaded into 0.7% agarose gel (pH = 7.4) in tris–
acetate EDTA (TAE) buffer containing 0.05 g/ml ethidium
lg) was used for these studies. The chalcone solution in phos-
c
D
l
c [28]. The calculations are based upon the published molar
absorptivities for ferricytochrome c (0.99 ꢂ 104 Mꢀ1 cmꢀ1) and fer-
rocytochrome c (2.99 ꢂ 104 Mꢀ1 cmꢀ1) at 550 nm.
l
bromide. The electrophoresis was carried out for 2 h at 50 V. After
electrophoresis, the gels were illuminated with UV light and pho-
tographed. The gel electrophoretic mobility of relaxed circle DNA
(form II) in agarose is about half that of supercoiled DNA (form I).
Enzymatic generation of superoxide anion radical in the pres-
ence of cytochrome c reductase (30 mg/ml), chalcones (200 lM)
and NADH (3 mM), in 50 mM phosphate buffer solution (pH =
7.4) was also followed by the reduction of ferricytochrome c.
2.10. Molecular modeling
2.6.2. EMR method
EMR spin trapping method was also used for the detection of
To assess the effects of –OCH3 substitutions in the phenyl ring
of chalcones (CH1–CH6) semi-empirical quantum mechanical
calculations were performed using Arguslab program version
4.0 running on windows workstation (www.arguslab.com). The
chalcones were geometry optimized using AM1 semi-empirical
method. The optimized structures were used to compute heat of
formation using AM1 Hamiltonian and properties of molecular
orbital and atomic charges using ZINDO/RHF (restricted Hartree–
Fock) semi-empirical Hamiltonian.
OÅ2ꢀ [29,30]. The reaction mixture (1 ml), containing chalcones
.
(200 lM) and DMPO (100 mM) in DMSO, placed in a quartz cuv-
ette, was continuously stirred during irradiation. The EMR micro-
wave power was 2 mW and modulation amplitude was kept at
0.5 G. Experiments were repeated to monitor the signal intensity
at different intervals of irradiation time. The transient radical spe-
cies were trapped by DMPO to form DMPO-adducts. The spectral
identification was confirmed by computer simulation using a
BASIC computer program. The output from this program was plot-
ted to get the simulated spectra.
3. Results and discussion
2.7. Cyclic voltammetry
3.1. Energy transfer process
Redox potentials of chalcones (1 mg; 5 ml acetonitrile) were
measured by cyclic voltammetry technique in a BAS 50A electro-
chemical analyzer (Bioanalytical systems, West Lafayette, IN) using
glassy carbon working electrode, platinum auxillary electrode and
Ag/AgCl reference electrode. Glassy carbon electrode was resur-
faced with alumina before use. Tetra-n-butylammonium perchlo-
rate (TBAP, 50 mM) was used as the supporting electrolyte. Each
solution was purged with nitrogen for 10 min prior to measure-
ment, and the cyclic voltammograms were recorded under nitro-
gen atmosphere. The redox potentials given are against Ag/AgCl,
unless otherwise mentioned.
The chemical structures of the chalcones are given in Fig. 1. RNO
bleaching assay was followed to measure the generation of 1O2.
The decrease of RNO absorbance at 440 nm as a function of irradi-
ation time for chalcones CH1, CH2, CH3, CH4, CH5 and CH6 is
shown in Fig. 2. The bleaching rate was used to calculate the 1O2
yield. The ratio of the slopes of RB to each chalcone was corrected
for molar absorption and photon energy to obtain the singlet oxy-
gen generation efficiency of the chalcones. 1O2 yields thus evalu-
ated are, 0.023, 0.034, 0.031, 0.038, 0.068 and 0.037 for CH1,
CH2, CH3, CH4, CH5 and CH6, respectively. To confirm the role of
1O2 in bleaching of RNO, experiments were carried out in the pres-
ence of specific singlet oxygen quencher, DABCO. The 1O2-quench-
ing rate constants of imidazole (10 mM, 2 ꢂ 107 Mꢀ1 sꢀ1) and
DABCO (10 mM, 1.5 ꢂ 107 Mꢀ1 sꢀ1) are comparable. Hence, the
rate of RNO bleaching should be reduced to 50% if equal concentra-
tion of imidazole and DABCO are used. Fig. 3 shows the loss of RNO
absorbance by CH2, CH5 and CH6 in the presence of DABCO, pro-
viding confirmative evidence for the photogeneration of 1O2.
Photogeneration of 1O2 by CH1, CH2, CH3, CH4, CH5 and CH6
was also monitored by the EMR-TEMPL method. When aerated
solutions of chalcones and TEMPL in DMSO were irradiated at
room temperature, EMR spectra consisting of three equally intense
lines, characteristic of a nitroxide radical, TEMPOL, were detected
and the EMR signal intensity was found to increase with increase
of irradiation time (Fig. 4). The effect of sodium azide (Fig. 4) on
the EMR signal intensity confirmed the 1O2 production. The photo-
generation of TEMPOL by chalcones was found to be parallel to
their 1O2 quantum yield, estimated by RNO bleaching method.
From Fig. 4, the 1O2 generating efficiencies (relative to RB) of
CH1, CH2, CH3, CH4, CH5 and CH6 were determined to be 0.03,
2.8. Antimicrobial activity: antifungal and antibacterial tests
Fresh solutions of sensitizers were prepared in DMSO and the
final concentration of DMSO was kept below 0.1%. Muller Hinton
agar medium was used to find out the antibacterial activity of com-
pounds against Gram-positive bacteria, Bacillus subtilis, and Gram-
negative bacteria, Escherichia coli. Chloramphenicol was used as
control. All selected species are fastidious microorganisms, safe
for experimentation. On the Muller Hinton agar plate 0.1 ml of log-
arithmic phase bacterial culture was inoculated. Well was pre-
pared on Muller Hinton agar plate using cork borer. Ten
microliter of each compound (10 mM) was placed in the corre-
sponding well and the plates were incubated at 30 °C for 24 h.
After 24 h, the plates were observed for antibacterial activity. The
activity was measured in terms of zone of inhibition against bacte-
ria (Gram-positive and Gram-negative) appearing around the well.
The assay was repeated three times and the average values were
recorded. The antifungal activity on yeast, Saccharomyces cerevisiae,
was also studied for the sensitizers using the above said procedure

Y. Yesuthangam et al. / Journal of Photochemistry and Photobiology B: Biology 102 (2011) 200–208
203
Fig. 1. Chemical structures of chalcones.
Time of Irradiation (min)
Time of Irradiation (min)
Fig. 2. Photosensitized RNO bleaching, measured at 440 nm in the presence of
imidazole (10 mM) in 50 mM phosphate buffer (pH = 7.4) with CH1 (}}}), CH2
(hhh), CH3 (+++), CH4 (ꢃꢃꢃ), CH5 (ooo) and CH6 (ꢄꢄꢄ), as a function of illumination
time.
Fig. 3. The effect of DABCO (10 mM) on the RNO bleaching, as measured by the
decrease in optical density at 440 nm by photosensitization with CH2 (hhh), CH5
(+++) and CH6 (ooo) in the presence of imidazole (10 mM) in 50 mM phosphate
buffer (pH = 7.4) as a function of illumination time. Photosensitized RNO bleaching
by CH2 (ꢃꢃꢃ), CH5 (ꢄꢄꢄ) and CH6 (}}}) in the absence of DABCO.
0.044, 0.04, 0.05, 0.08 and 0.048, respectively, which is comparable
with the values determined by the RNO bleaching method.
(pH = 7.4). The rates of cytochrome c reduction by chalcones CH1,
CH2, CH3, CH4, CH5 and CH6 were found to be 0.002, 0.004, 0.003,
3.2. Electron transfer process
0.004, 0.005 and 0.004 lM/s, respectively. The chalcones were
found to enhance the rate of cytochrome c reduction with different
efficiencies in the presence of electron donors such as EDTA, DETA-
PAC and NADH. Fig. 6 shows the effect of electron donors EDTA,
DETAPAC and NADH on the rates of superoxide generation (as
To detect the generation of OÅ2ꢀ, SOD inhibitable cytochrome c
reduction assay was followed. Fig. 5 shows the rate of cytochrome
c reduction when air saturated solutions of chalcones were photol-
ysed in the presence of cytochrome c (40
l
M) in phosphate buffer
measured by rate of cytochrome c reduction) by CH5. The

204
Y. Yesuthangam et al. / Journal of Photochemistry and Photobiology B: Biology 102 (2011) 200–208
Time of irradiation (sec)
Fig. 4. The formation of TEMPOL during photoirradiation of solutions containing RB
(hhh), CH5 (}}}), CH6 (ꢃꢃꢃ), CH2 (ꢄꢄꢄ) and CH1 (ooo) and in the presence of
TEMPL (20 mM) in DMSO. Inhibitory effect of 1 mM sodium azide ð/ / /Þ on the
intensity of TEMPOL radical during photoirradiation of CH5. The inset shows the
EPR spectra of TEMPOL generated during the photoirradiation of DMSO solution of
CH5 (1 mM) in the presence of TEMPL (20 mM) at 300 K: (A) 6-min irradiation, (B)
4-min irradiation, (C) 2-min irradiation and (D) in dark. Spectrometer settings:
microwave power, 2 mW; modulation frequency, 100 kHz; modulation amplitude,
0.5 G; time constant, 0.1 s; scan rate, 4 min; scan width, 200 G; and receiver gain,
500.
Fig. 6. The effect of EDTA (ꢄꢄꢄ), DETAPAC (}}}), NADH (ꢃꢃꢃ) and SOD (hhh) with
CH5 (ooo) on photosensitized superoxide generation measured as the rate of
cytochrome c reduction in the presence of ferricytochrome c (40
phosphate buffer, pH = 7.4, as a function of irradiation time.
lM) in 50 mM
Scheme 1. Photochemical pathways involved in the formation of ROS.
solution (Fig. 7B). The intensity of the EMR signal increased with
increase of irradiation time. The observed EMR spectrum could
be readily analyzed in terms of a mixture of two types of spin ad-
ducts. Hyperfine coupling constant (hfcc) values for one of the spin
adducts were simulated as a primary nitrogen triplet (AN = 13.8 G)
Time of irradiation (Sec)
Fig. 5. Photosensitized superoxide anion generation measured as the rate of
cytochrome c (40 lM) in 50 mM phosphate buffer (pH 7.4) with CH1 (+++), CH2
(hhh), CH3 (ꢃꢃꢃ), CH4 (ooo), CH5 (}}}) and CH6 (ꢄꢄꢄ) as a function of irradiation
ꢀ
ꢁ
split by a proton AbH ¼ 11:3 G , which is further split by a second-
time.
À
Á
ary proton AcH ¼ 1:3 G . Hyperfine coupling constants (hfccs) for
the second spin adduct are, AN = 14.4 G and Ab ¼ 14:3 G. These hfcc
H
enhanced rates are calculated to be 0.006, 0.009 and 0.012
l
M/s, in
values are in good agreement with the reported values for DMPO-
OÅ2ꢀ and DMPO-OH [32–35]. EMR spectra of these two spin adducts
were computer simulated separately using the corresponding hfcc
values. When these spectra were combined in the ratio of 8.5:1.5
for OÅ2ꢀ and ÅOH respectively, the simulated EMR spectrum
(Fig. 7C) matched well with the experimentally observed one.
the presence of EDTA, DETAPAC and NADH, respectively. The
enhanced photogeneration of OÅ2ꢀ in the presence of electron
donors indicates the involvement of anionic chalcone radical
intermediate [31]. As shown in Scheme 1, the interaction of
electron donors (D) with excited state chalcone (3S) leads to one-
electron reduction of chalcone to form anionic chalcone radical
(SÅꢀ). Interaction of this anion radical with molecular oxygen facil-
Addition of SOD (50
lg/ml) prior to irradiation inhibited the for-
mation of the adduct (Fig. 7D), confirming the generation of O2Åꢀ
.
itates the generation of O2Åꢀ
.
The reaction did not produce DMPO-CH3 adduct in the presence
of DMSO, indicating that no free ÅOH radical was generated. Forma-
tion of DMPO-OH adduct from DMPO-OOH adduct or a multi-step
process involving the trapping of chalcone (CH1) radicals could
possibly generate the DMPO-OH adduct. However, the absence of
EMR spin trapping experiment using DMPO as the spin trap was
also used to monitor the photogeneration of OÅ2ꢀ. A multiline EMR
spectrum was observed when CH1 (200
lM) was photolysed in
the presence of DMPO (100 mM) in an air-saturated DMSO

Y. Yesuthangam et al. / Journal of Photochemistry and Photobiology B: Biology 102 (2011) 200–208
205
Fig. 7. EMR spectrum obtained at room temperature in air-saturated DMSO
solution by irradiation of CH1 (100 M) and DMPO (100 M). (A) In the dark.
l
l
Fig. 8. EMR spectrum obtained at room temperature in air-saturated DMSO
solution by irradiation of CH5 (100 lM) and DMPO (100 lM). (A) In the dark. (B)
(B) After 4-min irradiation. (C) Computer-simulated spectrum, the spectrum
contains two spin adducts: DMPO-OÅ2ꢀ (AN ¼ 13:8 G; AHb ¼ 11:3 G; AcH ¼ 1:3 G) and
After 4-min irradiation. (C) Computer-simulated spectrum, the spectrum contains
DMPO-ÅOH adduct (AN = 14.4 G, Ab ¼ 14:3 G) in the ratio of 8.5:1.5. (D) In the
two spin adducts: DMPO-OÅ2ꢀ (AN = 13.2 G, AHb ¼ 10:5 G, AcH ¼ 1:4 G) and
H
DMPO-ÅOCH3 adduct (AN = 13.3 G, Ab ¼ 9:4 G and AcH ¼ 1:9 G) in the ratio of 8:2.
presence of SOD (50 lg/ml). Spectrometer settings are same as given in Fig. 4.
H
(D) In the presence of SOD (50
Fig. 4.
lg/ml). Spectrometer settings are same as given in
any characteristic hyperfine splitting pattern other than that of
ÅOH and OÅ2ꢀ adducts (Fig. 7) eliminates the involvement of
DMPO-CH1 radical adducts. Hence it is likely that the decomposi-
tion of DMPO-OOH adduct generated DMPO-OH adduct. However,
the DMPO-OOH adduct has been reported to be relatively more
stable in DMSO medium [36]. The observed decomposition of
DMPO-OOH could be due to the use of DMSO which may contain
traces of water.
3.3. Enzymatic generation of superoxide anion
The generation of superoxide anion radical by the enzymatic
reduction of chalcones CH1, CH2, CH3, CH4, CH5 and CH6, was
measured to be 0.009, 0.005, 0.0080, 0.004, 0.003 and 0.004 lM/s,
DMPO-spin trapping experiments of CH5 gave an EMR spec-
trum that could be readily analyzed in terms of a mixture of two
types of spin adducts. Hyperfine coupling constant (hfcc) values
for one of the spin adducts were arrived as AN = 13.2 G,
AbH ¼ 10:5 G and AcH ¼ 1:4 G. The hfccs of the other spin adducts
were, AN = 13.3 G, Ab ¼ 9:4 G and AcH ¼ 1:9 G. These spectra could
H
be readily assigned to DMPO-OÅ2ꢀ and DMPO-OCH3, respectively,
on the basis of reported hfcc values [32–35]. When the two spectra
were combined in the ratio of 8.0:2.0 for OÅ2ꢀ and OCH3 adducts,
Å
respectively, the simulated EMR spectrum (Fig. 8C) matched well
with the experimentally observed one. The mechanism for the for-
mation of DMPO-OCH3 adduct in the photoreaction of CH5 is not
clear. However, sacrificial loss of ÅOCH3 fragment by CH5 could ex-
plain the formation of DMPO-OCH3 adduct. The absence of any
characteristic hyperfine splitting pattern other than that of O2Åꢀ
Å
and OCH3 adducts eliminates the involvement of DMPO-CH5 rad-
ical adduct. Further, addition of SOD completely inhibited the for-
mation of any DMPO-radical adduct (Fig. 8D), suggesting that the
reaction of superoxide with chalcone or chalcone radical might
Time (sec)
Å
have generated the free OCH3 radical. Interestingly, the chalcone
Fig. 9. Enzymatic reduction of cytochrome c in the presence of chalcones (200
in 50 mM phosphate buffer (pH = 7.4) and in the presence of NADH and cytochrome
reductase. Control: cytochrome c + NADH + cytochrome reductase (hhh);
control + CH1
rrr); control + CH2 (ꢃꢃꢃ); control + CH3 (+++); control+CH4
(}}}); control + CH5 (ꢄꢄꢄ) and control + CH6 (ooo).
lM)
CH5 has highest number of –OCH3 group among the other candi-
dates, supporting the view of free OCH3 radical generation from
the chalcone. The other chalcones also showed similar type of
EMR spectra (data not shown).
Å
c
c
(

206
Y. Yesuthangam et al. / Journal of Photochemistry and Photobiology B: Biology 102 (2011) 200–208
Table 1
Table 3
ROS generation efficiency of chalcones CH1, CH2, CH3, CH4, CH5 and CH6.
Total energies, frontier orbital energies and electric dipole moments for CH1, CH2,
CH3, CH4, CH5 and CH6.
Chalcone
1O2 yielda
OÅ2ꢀ generation (
lM/s)
Compounds E1/2 (V)
PM3 (RHF)
(kcal/mol)
D
Hf
ZINDO (RHF)
Optical
method
EPR
method
Photo-
generation
Enzymatic
generation
HOMO
(au)a
LUMO
(au)
Dipole
moment
(debye)
CH1
CH2
CH3
CH4
CH5
CH6
0.023
0.034
0.031
0.038
0.068
0.037
0.030
0.044
0.040
0.050
0.080
0.048
0.002
0.004
0.003
0.004
0.005
0.004
0.009
0.005
0.008
0.004
0.003
0.004
CH1
CH2
CH3
CH4
CH5
CH6
ꢀ0.033
ꢀ0.054
ꢀ0.046
ꢀ0.170
ꢀ26.0903
ꢀ11.9944
ꢀ9.9459
ꢀ0.3130 ꢀ0.0285 5.4808
ꢀ0.2881 ꢀ0.0230 6.0708
ꢀ0.3138 ꢀ0.0238 5.8009
ꢀ0.3062 ꢀ0.0139 6.6558
ꢀ0.3031 ꢀ0.0085 6.9698
ꢀ0.3102 ꢀ0.0175 4.8911
ꢀ44.7294
a
ꢀ0.1825 ꢀ79.6970
Singlet oxygen yields are relative to RB.
ꢀ0.065
ꢀ46.5144
a
Atomic unit.
respectively (Fig. 9). Addition of SOD inhibited the cytochrome c
reduction, indicating that superoxide anion radical is involved in
the enzymatic reduction of cytochrome c [37,38]. The ROS genera-
tion efficiency of the chalcones is summarized in Table 1.
Table 4
Antimicrobial activity of chalcones CH1, CH2, CH3, CH4, CH5 and CH6.
3.4. Redox potentials
Chemical
Zone of inhibition (diameters in cm) against
compound
Electrochemical studies were carried out to determine the
reduction potential of the chalcones and the data are given in Table
2. All the chalcones showed one cathodic and one anodic peak.
The reduction potentials E1/2 for CH1, CH2, CH3, CH4, CH5 and
CH6 were found to be ꢀ0.033 V, ꢀ0.054 V, ꢀ0.046 V, ꢀ0.170 V,
ꢀ0.183 V and ꢀ0.065 V, respectively. The half wave potential of
CH5 is more negative than that of other chalcones. Electron-donat-
ing substituents are known to shift E1/2 to more negative value
[39]. The enzymatic reduction efficiencies of chalcones on cyto-
chrome c showed correlation with their reduction potentials.
Chalcone CH1 has less negative half wave potential than other
chalcones (Table 2) and it generates more superoxide anion radical.
Gram-positive
bacteriaa
Gram-negative
bacteriab
Yeast
cellc
CH1
CH2
CH3
CH4
CH5
CH6
No zone
No zone
No zone
No zone
No zone
No zone
0.14
0.08
0.11
0.05
0.04
0.06
0.07
0.04
No zone
No zone
No zone
No zone
a
b
c
Bacillus subtilis.
Escherichia coli.
Saccharomyces cerevisiae.
antifungal activity was found to be 0.07 and 0.04 cm in diameters,
respectively. Compound CH1 showed higher antifungal activity
compared to other chalcones. The biological activities can be com-
pared with the enzymatic superoxide anion generation efficiency
[42]. The higher antimicrobial activity of CH1 correlates with high-
er enzymatic superoxide anion generation in the dark. Chalcones
with higher E1/2 values showed better antimicrobial activity.
3.5. Electronic properties of chalcones
Results of molecular modeling of CH1, CH2, CH3, CH4, CH5 and
CH6 are presented in Table 3. The LUMO energies of the chalcones
are (Table 3) correlated well with experimental one-electron
reduction potentials, E1/2 [40,41]. In all the chalcones, the OH group
present in 20 position is in plane with the ring, but the bulky groups
attached with the propenone are projected out of plane, affecting
the co-planarity of the molecule.
3.7. Substituent effects on photodynamic generation of ROS
The chalcones investigated in this paper act as photosensitizers
and photodynamically generate ROS. The observed ROS generation
efficiency of the chalcones follows the order of CH5 > CH4 >
CH6 > CH2 > CH3 > CH1. This enhancement of photogeneration of
OÅ2ꢀ in the presence of electron donors is indicative of anionic prop-
erties of the radical intermediate formed during the photodynamic
process [31]. The radical formation may occur in the hydroxyl
group, attached to the phenyl ring of chalcones. The order of ROS
(1O2) generation efficiency of chalcones depends on the substitu-
ents attached to the phenyl group of the chalcone moiety. The
highest quantum yield of ROS by CH5 is due to the three eꢀ releas-
ing groups (methoxy) attached to the phenyl ring.
3.6. Biological activity
The in vitro antimicrobial activity of the chalcones was evalu-
ated against Gram-positive and Gram-negative bacteria and yeast
and their zones of inhibition against bacteria are found to be
0.14, 0.08, 0.11, 0.05, 0.04 and 0.06 cm for CH1, CH2, CH3, CH4,
CH5 and CH6, respectively (Table 4). Chalcone CH1 showed more
antibacterial activity than the other chalcones. No activity was
found with Gram-positive bacteria. Chalcones, CH1 and CH2
showed antifungal activity against yeast, S. cerevisiae, and their
Table 2
Cyclic voltammetric dataa for chalcones CH1, CH2, CH3, CH4, CH5 and CH6.
3.8. Photoinduced DNA cleavage
Substrate
Epc
Epa
D
Ep
E1/2
Chalcone mediated photocleavage of covalently closed circular
plasmid DNA was examined using CH4 and CH5 in phosphate buf-
fer (pH 7.4). Electrophorogram given in Fig. 10 shows photoin-
duced DNA cleavage by CH4 and CH5. Lane 1 contains DNA in
dark and Lane 2 has DNA with CH5 (1 mM) in dark and these lanes
serve as controls. Lanes 3 and 4 represent concentration-depen-
dent photocleavage by CH4. Lane 3 shows a decrease in the
CH1
CH2
CH3
CH4
CH5
CH6
ꢀ0.370
ꢀ0.278
ꢀ0.345
ꢀ0.663
ꢀ0.659
ꢀ0.400
0.304
0.171
0.254
0.323
0.294
0.271
0.674
0.449
0.599
0.986
0.953
0.671
ꢀ0.033
ꢀ0.054
ꢀ0.046
ꢀ0.170
ꢀ0.183
ꢀ0.065
a
Potentials in V against Ag/AgCl: scan rate: 100 mV/s.

Y. Yesuthangam et al. / Journal of Photochemistry and Photobiology B: Biology 102 (2011) 200–208
207
1
2
3
4
5
6
7
Form II
Form I
Fig. 10. Photoinduced scission of plasmid DNA (3 lg) mediated by CH4 and CH5 in phosphate buffer (pH 7.4) solution (irradiation time, 10 min). Lane 1: DNA alone in dark;
Lane 2: DNA + CH5 in dark; Lane 3: DNA + CH4 (2 mM); Lane 4: DNA + CH4 (1 mM); Lane 5: DNA + CH5 (2 mM); Lane 6: DNA + CH5 (1 mM); Lane 7: CH5 (2 mM) + SOD.
intensity of form I (supercoiled DNA) and the corresponding in-
crease in the intensity of form II (relaxed DNA) for higher concen-
tration of CH4 (2 mM). Compared to Lane 3, the intensity of form I
is higher in Lane 4 and the intensity of form II is reduced for lower
concentration of CH4 (1 mM). Similarly, lanes 5 and 6 show the ef-
fect of CH5 on the DNA scission. In both the lanes, compared to
Lane 1, decrease in the intensity of form I and increase in the inten-
sity of form II were observed. Thus, a concentration-dependent
DNA scission is observed for both CH4 and CH5 [43]. Comparison
of lanes 3 and 5 shows CH5 to exhibit enhanced activity than
and 1O2 (Type II) paths are involved in the photosensitization of
chalcones. The LUMO energies obtained by molecular modeling
correlate with the one-electron reduction potentials, measured
by cyclic voltammetry. Based on the results reported above, the ob-
served photoinduced DNA cleavage is due to the ROS generating
efficiency of the chalcones. It is also concluded that electron-
donating substituents directly attached to the photosensitizer
chalcones enhanced the ROS generation efficiency. Chalcones with
large number of electron donating (ring activating) groups may be
designed for enhanced photogeneration of ROS.
Åꢀ
Å
CH4. The major DNA-cleaving species are OH and H2O2, and O2
is relatively unreactive towards DNA [44]. However, the addition
of SOD protects plasmid DNA cleavage by the photosensitizer un-
der illumination (Fig. 10, Lane 7). This suggests the involvement
of superoxide-derived reactive oxygen species or superoxide med-
iated chalcone radical products in the DNA cleavage. Quantitative
analysis of band intensities was performed using computer pro-
gram written in Matlab software. The band intensities are repre-
sented in Fig. 11. These observations correlate well with the
higher ROS (1O2 and OÅ2ꢀ) generating efficiency of CH5 than CH4.
The role of ROS in the in vitro photoactivated DNA scission is sup-
ported by this investigation.
In conclusion, the synthetically prepared chalcones demon-
strated interesting properties of photogeneration of ROS. The enzy-
matic OÅ2ꢀ yield in the dark correlated with the E1/2. In general the
presence of more number of electron releasing –OCH3 groups en-
hances the photogenerating efficiency of ROS. Both OÅ2ꢀ (Type I)
Acknowledgements
We thank Prof. P. Sambasiva Rao, Pondicherry University, for
EMR spectral facility. Thanks are due to DST IRHPA for NMR facility
and UGC, New Delhi, for the award of teacher fellowship to Y.
Yesuthangam.
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